AU2022331118A1 - Burden-addicted production strains - Google Patents

Abstract

The invention provides a microbial production cell for synthesis of a product, further comprising a burden-addiction genetic circuit whose expression confers a selective growth and/or survival advantage on those cells that synthesize the product; while limiting proliferation of low- or non-productive escaper cells.

Description

TITLE: Burden-addicted production strains

FIELD OF THE INVENTION

The invention provides a microbial production cell for synthesis of a product, further comprising a burden-addiction genetic circuit whose expression confers a selective growth and/or survival advantage on those cells that synthesize the product; while limiting proliferation of non/low-productive escaper cells.

BACKGROUND OF THE INVENTION

An increasing share of the world's chemical production relies on microorganisms that are genetically engineered to function as cell factories, and tailor-made for the biosynthesis of a given molecule. Production processes, employing these cell-factories, are typically initiated from a starter culture of a small number of cells of a production organism, which go through a phase of growth and expansion of cell numbers in large fermentation tanks (up to 1,000,000 L volume). In some setups, production of a given molecule proceeds both during the growth phase and during a subsequent period (batch and fed-batch cultures); and alternatively, production may be continuous. A chemostat fermenter allows a production organism to be grown in a fermentation broth that is constantly diluted, thus tapping product and cells from the culture, while replenishing with fresh nutrient medium. On an industrial scale, such production processes may continue operation for 1-2 months before starting a new culture in a clean tank. The fermentation processes and equipment used in this industry are very similar, both for the production of a wide range of commodity small molecules and for therapeutic proteins, and consequently these processes are subject to similar problems. In other applications, microbial cells may be engineered to produce a non-native molecule that confers a commercial advantage in the environment in which the cell is cultured.

Cell factories engineered to allocate finite metabolic resources to the biosynthesis of a given molecule are subject of an unnatural load; commonly reflected in slower growth. This load places a selective pressure for cells unable to synthesize the product molecule (non-producing or lower-producing), this problem particularly arising when the production run is for extended periods of time (e.g. in chemostats). Such non-producing cells within an industrial fermentation are highly undesirable, since they consume nutrients, oxygen and space. Since lower- and non-producing cells grow faster, they have a strong selective advantage over producing cells. In a growing cell culture, such improvements in fitness can lead to significant out-competition of the producing cells over time. This drift from the optimal production state is an eventual reason for discarding the fermentation broth and spending resources on cleaning, sterilization, not to mention nutrients, to replenish the fermentation tank with new, producing organisms. Such non-producing cells originate from genetic mutations that arise in the cells of an original producing organism undergoing many growth divisions.

Since the occurrence of genetic mutations and non-genetic adaptation in cells of a production organism that lead to a decline in product formation by the cells during a production run, cannot be avoided, there is a need for methods for eliminating or slowing the growth of non-producing cells in the production. Preferably, such methods of elimination are sufficiently effective that they prevent the observed drift from the production state, and thereby prolong the life-time of an industrial fermentation.

The present invention addresses the problem of how to deprive non-producing members of a cell factory with a fitness advantage; such as to delay their proliferation amongst members of a productive cell factory over time, and thereby improve the productivity of the cell factory.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a microbial production cell genetically engineered to synthesize a product, wherein said cell further comprises:

- a first essential gene operably linked to a first burden-sensing promoter, and

- a second essential gene operably linked to a second burden-sensing promoter wherein said first burden-sensing promoter is heterologous with respect to said first essential gene, and said second burden-sensing promoter is heterologous with respect to said second essential gene; wherein synthesis of the product confers a burden and/or fitness cost on said cell; and wherein expression of said first essential gene is up- regulated when said first burden-sensing promoter is induced by said burden and/or fitness cost relative to a basal level expression of said essential gene when said first burden-sensing promoter is not induced, and expression of said second essential genes is up-regulated when said second burden-sensing promoter is induced by said burden and /or fitness cost relative to a basal level expression of said second essential gene when said second burden-sensing promoter is not induced.

A second aspect of the invention provides a method of product biosynthesis comprising the steps of:

- providing at least one microbial production cell of the invention, - introducing the at least one cell into a cultivation medium comprising substrate for production of said product,

- recovering said product.

A third aspect of the invention provides for a use of a first and a second essential gene operably linked to a first and a second burden-sensing promoter, respectively, to enhance product yield of a cultured population of microbial production cells arising from a single cell following at least 50 generations; wherein said first burden-sensing promoter is heterologous with respect to said first essential gene, and said second burden-sensing promoter is heterologous with respect to said second essential gene; wherein production of the product confers a burden on said cell; and wherein expression of said first essential gene is up-regulated when said first burden-sensing promoter is induced by said burden relative to a basal level expression of said first essential gene when said first burden-sensing promoter is not induced, and expression of said second essential gene is up-regulated when said second burden-sensing promoter is induced by said burden relative to a basal level expression of said second essential gene when said second burden-sensing promoter is not induced.

A fourth aspect of the invention provides for a use of a microbial production cell of the invention for producing a biosynthetic product.

A further aspect of the invention provides a microbial production cell genetically engineered to synthesize a product, wherein said cell further comprises:

- an essential gene operably linked to a burden-sensing promoter wherein said burden-sensing promoter is heterologous with respect to said essential gene; wherein synthesis of the product confers a burden and/or fitness cost on said cell; wherein expression of said essential gene is up-regulated when said burden-sensing promoter is induced by said burden and/or fitness cost relative to a basal level expression of said essential gene when said burden-sensing promoter is not induced; and wherein said microbial production cell belongs to a genus selected from among Bacillus, Corynebacterium, and Aspergillus. DESCRIPTION OF THE INVENTION Brief description of the figures: Figure 1: Graph showing growth (measured as optical density: OD 630nm) over time of cells of the parent strain E. coli BL21(DE3) (grey line) and cells of the parent strain transformed with a hGH-GFP production plasmid pEG34 (black line; pEG34). Growth of four replicate cultures of each strain was measured under hGH-GFP expression inducing conditions; error bars depict standard error of the mean (n = 4). Figure 2: Graph showing growth (measured as optical density: OD 630nm) over time of cells of the parent strain E. coli BL21(DE3) (black line) and cells of the non-producing burden-addicted E. coli strains s5.0#3, s7.0#8 and s9.0#8 derived from the parent strain (as indicated; wherein essential genes folP-glmM are controlled by p_yccV, p_ycjX and pmutM respectively). The measured growth rate is based on an average of four replicate cultures (n = 4). Figure 3: Graph showing growth (measured as optical density: OD 630_nm) over time of cells of the burden-addicted strain s9.0#8 harboring either the hGH-GFP production plasmid pEG34 (black line) or the control plasmid pEG0 (grey line). Growth of four replicate cultures of each strain was measured under hGH-GFP expression inducing conditions; error bars depict standard error of the mean (n = 4). Figure 4: Graph showing growth (measured as optical density: OD 630_nm) over time of cells of the parent strain E. coli BL21(DE3) (black line), and cells of three burden- addicted strains: s5.0#3, s7.0#8 and s9.0#8 harboring and expressing the hGH-GFP production plasmid pEG34 (as indicated). The measured growth rate, measured under hGH-GFP expression inducing conditions, is based on an average of four replicate cultures (n = 4). Figure 5: Histogram of GFP/OD values (Y-axis) measured during the first and sixth culture transfer (seed 1 and 6) of cultures of the hGH-GFP producing strains: s3.6 (p_ibpA), s6.6 (p_grpE), s7.6 (pycjX), and s10.6 (pybbN) comprising the respective burden-sensing promoters and one of four variant RBS coding sequences, driving expression of the essential gene, folP-glmM. Cultures of non-burden addicted E. coli strain BEG34 and the parent strain E. coli BL21(DE3), BEG0, are included as controls. Measurements are performed on cultures grown under hGH-GFP expression inducing conditions. Error bars depict standard error of the mean (n = 4). Figure 6: Histogram of GFP/OD values (Y-axis) measured after serial culture transfers (seed 1 to 12) of cultures of the hGH-GFP producing strains: s3.6#2 (pibpA), s6.6#6 (p_grpE), s7.6#8 (p_ycjX), and s10.6#7 (pybbN) comprising the respective burden-sensing promoters and one of four variant RBS coding sequences, driving expression of the essential gene, folP-glmM. Cultures of non-burden addicted E. coli strain BEG34 and the parent strain E. coli BL21(DE3) are included as controls. Measurements are performed on cultures grown under hGH-GFP expression inducing conditions. Error bars depict standard error of the mean (n = 3). Figure 7: Histogram of OD/min as a measure of the growth rate of burden-addicted hGH-GFP producing strains s3.6#2, s6.6#6, s7.6#8 and s10.6#7 compared to non- burden addicted hGH-GFP producing E. coli strain BEG34 and the parent strain E. coli BL21-DE3 (BEG0). The growth rate of the respective strains is based on 7 replicate cultures measured under hGH-GFP expression inducing conditions at seed 0; error bars denote standard error of the mean (n = 7). Figure 8: Graphs showing GFP/OD values (Y-axis) measured after serial culture transfers (seed 1, 4 to 9) of cultures of hGH-GFP producing strains, minus the values measured for an empty plasmid control BEG0-empty strain. The strains shown in: (A) s7.6#8 (pycjX), (B) s13.6#2 (pfsxA), and (C) s13.6#2evo (pfsxA), each comprise the respective burden-sensing promoter driving expression of the essential gene, folP-glmM (gray line) and a non-burden addicted hGH-GFP producing E. coli strain BEG34 (black line). Measurements are performed on cultures grown under hGH-GFP expression inducing conditions. Error bars depict standard error of the mean (n = 5). Figure 9: Graphs showing GFP/OD values (Y-axis) measured after serial culture transfers (seed 1, 4 to 9) of cultures of hGH-GFP producing strains, minus the values measured for an empty plasmid control BEG0-empty strain. The strains shown in: (A) s15.6.7 (p_rrnB) and (B) s16.6.6 (p_rrnE), each comprise the respective burden-sensing promoter driving expression of the essential gene, folP-glmM (gray line) and a non- burden addicted hGH-GFP producing E. coli strain BEG34 (black line). Measurements are performed on cultures grown under hGH-GFP expression inducing conditions. Error bars depict standard error of the mean (n = 5). Figure 10: Graph showing relative hGH-GFP production (Y-axis) measured after serial culture transfers (seed 2 to 5, and 7) of cultures of a hGH-GFP producing strain, s29.6#3, minus the values measured for a parallel empty plasmid control BEG0 strain. Strain s29.6#3 comprises the osmotic stress sensing promoter ppoxB driving expression of the essential gene, folP-glmM (black squares), compared to non-burden addicted hGH-GFP producing E. coli strain BEG34 (gray dots). Serial passage 7 was analyzed after 72 h. Measurements are performed on cultures grown under hGH-GFP expression inducing conditions. Error bars depict standard error of the mean (n = 5 for s29.6#3 and n = 8 for BEG34).

Figure 11: Histogram showing S. aureus lysis rate per minute, normalized to E. coli culture OD, as a measure of the secreted lysostaphin production rate of cultures of lysostaphin-producing strains s6.4#5 comprising the burden sensing promoter p_grPE and one of four variant RBS coding sequences driving expression of the essential gene, folP- glmM, compared to non-burden addicted lysostaphin producing E. coli strain BENDU5cam. The rate was measured on cultures grown under lysostaphin expression inducing conditions after serial culture transfers (seed 1 and 7). Error bars depict standard error of the mean (n = 3).

Figure 12: Graph showing mevalonic acid titer (g/L) synthesized by cultures of the burden-addicted mevalonic acid-producing strains S19.1# 1(P_CS_PD) and s9.1.4(p_mutM), measured after serial culture transfers (seed 2, 5 and 6) compared to the parent nonburden addicted mevalonic acid-producing E. coli strain BL21(DE3)-pMevT. The burden sensing promoters, p_csPD and p_mutM, are each combined with one of four variant RBS coding sequences driving expression of the essential gene, folP-glmM. Measurements are performed on cultures grown under MevT expression inducing conditions. Error bars depict standard error of the mean (n = 3).

Figure 13: Bar plot showing human serum albumin (hSA) production titers of cultures of Pichia pastoris strain EGS31 comprising a genomically-integrated copy of a cDNA version of the hSA coding ALBI gene under control of the AOX1 promoter, and a PDI1 promoter operably linked to the kanMX selection gene. The cultures were grown under either selecting conditions (addiction activated by 750 |ig/mL G418) or non-selecting conditions (addiction not activated). The hSA titer of the 'addiction not activated' culture is set to 100%, and the titer of the 'addiction activated culture is shown as a percent thereof.

Figure 14 A and B: Bar plot showing human serum albumin (hSA) production titers of cultures of burden-addicted versions of the Pichia pastoris strain EGS31 each comprising a genomically-integrated copy of a cDNA version of the hSA coding ALBI gene under control of the AOX1 promoter and a kanMX selection gene operably fused to a burden responsive promoter FPR2, or RPL3 or RPL6A. The cultures were grown under either non-selecting conditions (addiction not activated) or under selecting conditions (addiction activated with either 150 or 750 |ig/mL G418) for a period of about 30 cell divisions ([A] Seed 1), or after a further 10 cell divisions ([ B] Seed 2). Average hSA titers are shown in percent of the titer in the seed 1 'EGS31' culture. Error bars depict standard error of the mean (n = 4).

Figure 15: Comparison of IgA-GFP production of burden-addicted EGS340 and parental EGS84 in long-term cultivation. Demonstration of burden addiction in Bacillus to sustain production using the IgA production burden-sensing perR promoter to regulate transcription of the essential IscU gene operon (sufC-sufD-sufS-sufU-sufB'). Comparison in EGS340 and EGS84 during serial-passaging experiment at 30 deg. C in which a 500x dilution seed is transferred to new culture medium every 24 hours. Points are averages and error bars show s.e.m. (n = 7).

Figure 16: Comparison of EGS343 and EGS84 in long-term cultivation. Demonstration of burden addiction in Bacillus subtilis to sustain production using the perR promoter to regulate transcription of the essential accC gene operon (accB-accC-yhqY). Comparison of IgA-GFP production in EGS343 and EGS84 during serial-passaging experiment at 30 deg. C in which a 500x dilution seed is transferred to new culture medium every 24 hours. Points are averages and error bars show s.e.m. (n = 7).

Figure 17: Use of several burden-sensors to simultaneously regulate different essential genes and thereby sustain production of a heterologous protein in Bacillus subtilis. Comparison of IgA-GFP production strains following five serial passages (500x dilution seeds) corresponding to approximately 75 generations. The single burdensensor strain EGS340 carries only the p_perR-based burden sensor controlling transcription of the IscU essential gene operon (EGS340). EGS340 is compared to derived strains in which transcription of the accC essential gene operon is also regulated by respectively a p_ctsR-based burden sensor (EGS460), a pdnaK-based burden sensor (EGS462), and a p_hrcA-based burden sensor (EGS466). Bars show averages and error bars show s.e.m. (n = 4).

Figure 18: Comparison of relative IgA-GFP levels measured by IgA specific ELISA following approx. 75 generations of cell cultivations in the non-burden addicted control strain EGS084 and the double-burden addicted derived strain EGS466 (based on a PperR controlling the IscU essential gene operon and a phrcA controlling the accC essential gene operon).

Figure 19: Use of TIS variation on an essential gene to regulate growth rate. Two biological replicates of the parent strain (EGS621) and the strains with the TIS sequence variations of the essential gene CIA1 (EGS1100, EGS1101, EGS1102, EGS1104) grown in YPD. The Pichia strains exhibited growth correlated to the relative strength of the four TIS sequences.

Abbreviations, terms and definitions:

Burden refers to the cellular state of a microbial cell (such as a cell genetically engineered to synthesize a product)during production of a product under production conditions, and which results in a fitness cost attributable to the synthesis of said product, in particular in a cell that exhibits high level efficient synthesis of the product. Fitness cost is a measure of said burden that can be quantified by measuring the percent reduction in the maximum exponential growth rate of the cell (measured along the growth curve) during synthesis of the product under production conditions as compared to a parent microbial cell incapable of said synthesis when grown under comparable production conditions.

Burden-sensing promoter refers to a promoter that is induced by said 'burden', and when induced, said burden-sensing promoter can upregulate the expression of a gene to which it is operably linked in a microbial production cell, when cultured under production conditions as compared to a mutant derivative of said microbial production cell that synthesizes essentially none, or at least 50 % less, of the intended product of the production cell. Such mutant derivative includes a non-productive escape mutant isolated following long-term cultivation of a population of cells derived from said production cell. Non-limiting examples (see Table 2 for details) of burden-sensing promoters include promoters of the E. coli genes htpG, ibpA, dpB, yccV, grpE, ycjX, IdhA, mutM, ybbN, prIC, groES, fxsA, htpX, rrnB, rrnE, cspD, katE, xthA, uspE, gadB, ahpC, katG, grxA, oxyS, poxB, trxC and their homolog versions in other gram-positive and gram-negative bacteria; for S. cerevisiae non-limiting examples of burden-sensing promoters include promoter of the genes KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGGI, RAD51, RAD54; for Bacillus non-limiting examples of burden-sensing promoters include promoter of the genes groES, ctsR, dnaK, perR, hrcA, spx, sigB, yfIT and their homolog versions; for Corynebacterium non-limiting examples of burden-sensing promoters include promoter of the genes groES, kata, cpIX, mutM and their homolog versions; for Aspergillus non-limiting examples of burden-sensing promoters include promoter of the genes bipA, dxA and their homolog versions. Burden-sensing promoters may also be hybrids, scrambled or truncated versions of such natural promoters as long as such promoters still maintain the response to burden. Burden-addicted microbial cell refers to a microbial cell engineered to comprise a genetic circuit comprising at least one 'burden-sensing promoter' operably linked to an essential gene of the cell. Preferably said cell has a genetic circuit comprising two, namely a first essential gene operably linked to a first burden-sensing promoter, and a second essential gene operably linked to a second burden-sensing promoter wherein said first burden-sensing promoter is heterologous with respect to said first essential gene, and said second burden-sensing promoter is heterologous with respect to said second essential gene

Burden-addicted microbial production cell refers to microbial cell genetically engineered to synthesize a desired product, said cell being further genetically engineered to comprise a genetic circuit comprising said at least one, preferably two, 'burden-sensing promoter' operably linked to an 'essential gene' of the cell, as defined above. The desired product may be a protein encoded by a nucleic acid molecule in said engineered microbial cell, or a product of a heterologous pathway encoded by one or more nucleic acid molecules to be expressed by the cell.

Non-burden addicted microbial production cell is considered to be a "parent" cell of the burden addicted microbial production cell. It is a microbial production cell comprising and expressing one or more genes encoding the product or encoding a metabolic pathway for biosynthesis of the product, but lacking the genetic circuit comprising said at least one, preferably two, burden-sensing promoter operably linked to an essential gene as disclosed in the present invention. The at least one, preferably two, essential genes in this non-burden addicted microbial production cell wherein each is operably linked to its native promoter.

Desired products of the invention include - but are not limited to - a product that, when synthesized by the genetically engineered microbial production cell, conveys a fitness cost (burden) upon the production cell. Non-limiting examples of such product include an organic acid, terpenoid, isoprenoid, polyketide, alcohol, sugar, vitamin, aldehyde, carboxylic acid, fatty acid, amino acid, peptide, enzyme, therapeutic protein and precursors thereof, such as human growth hormone, insulin, glucagon-like peptide- 1, a monoclonal or polyclonal antibody, a single-fragment antibody, and a nanobody. Examples further include proteins naturally found in eggs such as ovalbumin, or milk such as casein, lactadherin, alpha-lact lactoferrin, secreted immunoglobulin A and G, a secretory component. Examples of an enzyme includes an amylase, lipase, protease, barnase, p-galactosidase, crystal protein, cutinase, PETase, and laccase as well as a carbohydrate active enzyme such as a xylanase, lichenase, cellulase, lytic polysaccharide monooxoygenase, and a pectase. Essential gene (or essential gene operon) refers to gene(s) in the microbial production cell which, if down-regulated, lead to reduction in the growth rate of the microbial production cell under production conditions. Preferably an essential gene is essential for growth irrespective of the nutrient composition of these production conditions, whereby sufficient expression of such essential genes to support cell growth would not be dependent on the presence or absence of specific inhibitors or nutrients provided under the production conditions . Non-limiting examples of E. coll essential genes during standard laboratory conditions include folP-glmM, glmM, murl, asd, thyA, rpoD, nusG, rpsU, accD, degS, fldA, ftsN, hfIB, lolA, mraY, mreD, murA, murB, murF, nadD, rpIV, rpsG, and homologues thereof. Non-limiting examples for Bacillus include IscU operon (sufC-sufD-sufS-sufU-sufB), accC operon (accB-accC-yhqY), glmM, ylaN, InfA, dapA and homologues thereof. Non-limiting examples for Saccharomyces include, FOL1, MED7, RRP40, NOP8, PGI1, NEP1, URA3 LEU2, TRP1, HIS3 and homologues thereof. Nonlimiting examples for Aspergillus include ARG3, adeA, and homologues thereof: Nonlimiting examples for Corynebacterium include air, glmM, and homologues thereof: For example, suitable genes encode enzymes responsible for synthesizing cell wall constituents. Further, an essential gene used in the context of this invention preferably neither encodes a desired product to be expressed by the production strain; nor encodes a protein that facilitates synthesis of a desired product or intermediate thereof in a heterologous pathway to be expressed by the production strain.

Conditional essential gene is one that allows the burden addiction system in the microbial production cell to be "activated" by culturing the cells under the condition of essentiality such as added antibiotic or removed nutrient.

Essential gene basal expression level is the level of transcription of each 'essential gene' (as defined above) that is operably linked to a 'burden-sensing promoter' in a burden-addicted microbial production cell, when the 'burden-sensing promoter' is not induced. The burden sensing promoter to which the essential gene is operably linked is heterologous with respect to said essential gene, in the sense that the promoter is not the native promoter of said essential gene (even though the promoter may be present in the same genome), and is thus not found operably linked to said essential gene in nature. Said basal (i.e. un-induced) level expression of an essential gene is a level sufficient to support growth of a cell under production conditions (or during an exponential growth phase) at a level equal to or less than 10, 20, 50, 90 or 95% of the growth rate of a corresponding cell wherein each said essential gene is operably linked to its native promoter. A lowered growth rate due to basal level essential gene expression in those cells where the burden sensing promoter is not activated constitutes a selective disadvantage for non-productive cells, for example non-productive mutants arising during product production.

Fitness cost: is quantified by comparing the maximum exponential growth rate (when measured along the growth curve) of a non-burden addicted microbial production cell or cell culture thereof (i.e. lacking burden-sensing promoters operably linked to the at least one, preferably two essential genes), but comprising and expressing one or more genes encoding the product or encoding a metabolic pathway for biosynthesis of the product) relative to the maximum exponential growth rate of (i) a parent microbial cell (or cell culture thereof) either devoid or incapable of expressing gene(s) encoding the product or metabolic pathway for biosynthesis of the product, from which the microbial production cell was derived (or (ii) an escape cell derived from the non-burden addicted microbial production cell, wherein said escape cell produces less than 50% of the product compared to the non-burdened addicted microbial production cell), when grown under comparable production conditions. The maximum exponential growth rate of the respective cells or cell cultures thereof is to be measured at the start of production following introduction of the respective cells or cell cultures thereof into a cultivation medium comprising substrate for production of said product.

Production conditions refers to specific cultivation conditions to be used during production and can relate to medium and/or other culture conditions (e.g. temperature, pH, stirring, aeration, etc) for production of a desired product by the microbial production cell.

Ribosomal binding site (RBS), translation initiation region, or translational strength element refers to the genetic region of the 5' untranslated region that control the translation strength of a particular messenger RNA.

Detailed description of the invention:

I. A burden-addicted microbial production cell

A first aspect of the present invention provides a microbial production cell genetically engineered to synthesize a product, wherein said cell further comprises: a. an essential gene operably linked to a burden-sensing promoter, wherein said promoter is heterologous with respect to said essential gene; wherein production of the product confers a burden and/or fitness cost on said cell, and wherein expression of said essential gene is up-regulated when said burden-sensing promoter is induced by said burden and/or fitness cost relative to a basal level expression of said essential gene when said burden-sensing promoter is not induced.

In a further aspect of the present invention, the microbial production cell genetically engineered to synthesize a product comprises: a. a first essential gene operably linked to a first burden-sensing promoter, and b. a second essential gene operatively linked to a second burden-sensing promoter, wherein said first burden-sensing promoters is heterologous with respect to said first essential genes, and said second burden-sensing promoters is heterologous with respect to said second essential genes; wherein synthesis of the product confers a burden on said cell, and wherein expression of said first essential genes is up-regulated when said first burdensensing promoter is induced by said burden relative to a basal level expression of said first essential gene when said first burden-sensing promoter is not induced, and expression of said second essential genes is up-regulated when said second burdensensing promoter is induced by said burden relative to a basal level expression of said second essential gene when said second burden-sensing promoter is not induced.

In one embodiment, the first and second essential genes are different. In another embodiment, the first and second essential genes are identical. In a specific embodiment, the first and second essential genes are identical while the first and second burden-sensing promoters are different.

In one embodiment, the first and second burden-sensing promoters are different. In another embodiment, the first and second burden-sensing promoters are identical. In a specific embodiment, the first and second burden-sensing promoters are identical while the first and second essential genes are different.

In one embodiment, the microbial production cell comprises more than two essential gene operably each linked to a burden-sensing promoter. In one embodiment, the invention provides a microbial production cell genetically engineered to synthesize a product, said microbial cell comprising two, three, four, five, six or more burden sensing promoters to control essential genes in the cell.

In one embodiment, the microbial cell comprises three essential genes, each operably linked to a burden-sensing promoter. In one embodiment, the microbial cell comprises four essential genes, each operably linked to a burden-sensing promoters. In one embodiment, all the burden-sensing promoters are different. In another embodiment, some of the burden-sensing promoters are identical, but they are linked to different essential genes. In one embodiment, all the essential genes are different. In another embodiment, some of the essential genes are identical, but they are linked to different burden-sensing promoters.

A microbial production cell genetically engineered to synthesize a product is one that comprises one or more genes encoding the product or encoding a metabolic pathway for synthesis of the product, wherein optionally said one or more genes are operably linked to a constitutive or inducible promoter. In one embodiment, the one or more genes encoding the product or encoding a metabolic pathway for synthesis of the product may be recombinant.

According to one embodiment, said burden and/or fitness cost on said microbial production cell of the invention, is quantified by comparing the maximum exponential growth rate of the microbial production cell comprising one or more genes encoding the product or encoding a metabolic pathway for synthesis of the product, (but lacking burden-sensing promoters operably linked to the at least one preferably two essential gene), relative to the maximum exponential growth rate of a parent microbial cell lacking or incapable of expressing one or more genes encoding the product or encoding a metabolic pathway for synthesis of the product, and from which said microbial production cell was derived, where the respective cells are cultured under essentially identical production conditions. The burden and/or fitness cost on said microbial production cell preferably corresponds to a percent reduction in the quantified maximum exponential growth rate selected from among > 5 %, >10%, >15%, >20%, >25%, >35% and > 45 %.

The burden-addicted microbial production cell according to the invention may be any prokaryotic or eukaryotic microorganism such as a bacterium, yeast, and filamentous fungus.

In one embodiment, the microbial production cell of the invention is a prokaryote. A non-exhaustive list of suitable bacteria is given as follows: a species belonging to the genus selected from among Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Proprionibacterium, Bacteroides and Bifidobacterium.

In one preferred embodiment, the invention provides a microbial production cell genetically engineered to synthesize a product, said cell comprising an essential gene operably linked to a burden-sensing promoter; wherein said microbial production cell belongs to the genus Bacillus.In another embodiment, the microbial production cell of the invention is a eukaryote, such as a yeast or fungus. In certain embodiments, the eukaryote can be a member of the genus Saccharomyces, Komagataella or Aspergillus.

A non-exhaustive list of suitable yeasts is given as follows: a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum; a yeast belonging to the genus Kluyveromyces, e.g. K. lactis K. marxianus var. marxianus, K. thermotolerans; a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis; a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, or other yeast genera, e.g. Cryptococcus, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces or Schizosaccharomyces.

A non-exhaustive list of suitable filamentous fungi is given as follows: a filamentous fungus belonging to the genus of Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma Thermomyces, Streptomyces and Aspergillus. More specifically, the filamentous fungus may be selected from Fusarium oxysporum, A. niger, A. awamori, A. oryzae, and A. nidulans.

The product(s) synthesized by the microbial production cell of the invention is one that incurs a burden on the cell and fitness cost reflected in a reduction in growth rate, such product(s) including amino acids, organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, peptides, enzymes, therapeutic proteins and their precursors such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, single-fragment antibodies and nanobodies. Examples further include proteins naturally found in eggs such as ovalbumin, or milk such as caseins, lactadherins, alpha-lactalbumin, betalactoglobulin, osteopontin, lactoferrin, secreted immunoglobulin A and G, secretory components. Further examples of enzymes are amylases, lipases, proteases, barnases, p-galactosidases, crystal proteins, cutinases, PETases, and laccases as well as carbohydrate-active enzymes such as xylanases, lichenases, cellulases, lytic polysaccharide monooxoygenases, and pectases. In one embodiment the product is not a native product of the microbial production cell of the invention, and thus not produced by a parent cell from which the microbial production cell was derived. In one embodiment, the one or more genes encoding the product or encoding a metabolic pathway for synthesis of the product are heterologous with respect to the microbial production cell of the invention; where said one or more genes may be transgenes. II. Features of the microbial production cell of the invention

II. i Fitness cost - production burden

Heterologous expression of a desired product or pathways leading to a desired product in a microbial cell imposes a burden on the cell. Burden can also result from overproduction of a native product, e.g. in cells of microbial strains selected following mutagenesis. The burden may be described as a fitness cost which results in slower growth of the cell (Example 1; figure 1). This may be due to general and/or productspecific metabolic toxicities, increased use and/or depletion of potentially finite cell resources and metabolites, which a microbial cell experiences during the biosynthesis of a product of the invention under production conditions. Particularly during industrial scale fermentation, a combination of fitness cost and slower growth places a selective pressure on the population of cells in the fermenter, which over time leads to a decline in product yield due to an accumulation of non-productive cells resulting from genetic mutations or non-genetic variation (example 2, BEG34 in figure 6).

In the present invention, the presence and expression of one or more genes encoding a product or encoding a metabolic pathway for biosynthesis of a product in a microbial production cell confers a fitness cost (burden).

The fitness cost of producing a product on a microbial production cell is quantified by comparing the growth rate of a non-burden addicted microbial production cell (i.e. lacking at least one, preferably two, burden-sensing promoters operably linked to the essential genes, but comprising and expressing one or more genes encoding the product or encoding a metabolic pathway for biosynthesis of the product) relative to the growth rate of (i) a parent microbial cell from which the non-burden addicted microbial production cell was derived, wherein said parent microbial cells is devoid of gene(s) encoding the product or metabolic pathway for biosynthesis of the product, or (ii) an escape cell derived from the non-burden addicted microbial production cell, wherein said escape cell produces less than 50% of the product compared to the non-burdened addicted microbial production cell. The burden and/or fitness cost on said microbial production cell preferably corresponds to a percent reduction in the quantified growth rate selected from among > 5 %, >10%, >15%, >20%, >25%, >35% and >45%, more preferably at least 5%.

Preferably quantification of the relative growth rates is performed on the respective microbial cells by measuring their maximum exponential growth rate when cultured under essentially identical conditions, these conditions being chosen to closely mimic those in which eventual large-scale fermentation is to take place. All uses of growth rate refers to the term specific growth rate.

II. II Essential gene

A microbial production cell of the invention comprises at least one preferably two essential gene(s) encoding at least one preferably two protein(s), respectively, whose expression is required for cell growth and/or survival.

In one embodiment, the microbial production cell comprises a first and a second essential gene encoding a first and a second protein (where the first and second protein may be the same or different), respectively, wherein the expression of both said first and said second protein is required for cell growth and/or survival.

Preferably, the at least one preferably two essential genes and their expression do not indirectly cause a fall in production of the desired product by the production cell. Further, the essential gene(s) used in the context of this invention do not encode or lead to synthesis of a desired product or intermediate of a heterologous pathway to be expressed in the microbial production cell.

The at least one preferably two essential gene(s) are preferably non-conditional essential gene(s), such that expression of the gene(s) is essential for cell growth and/or survival irrespective of the composition of the growth medium or conditions in which the cell is cultured.

In one embodiment of the invention, when the production cell is a prokaryote, such as E. coll, the at least one preferably two non-conditional essential gene(s) are selected from folP-glmM, glmM, murl, asd, thyA, usA, rpoD, nusG, rpsU, accD, degS, fldA, ftsN, hfIB, lolA, mraY, mreD, murA, murB, murF, nadD, rpIV and rpsG, and homologues thereof.

In a further embodiment of the invention, when the production cell is a prokaryote, such as a Bacillus strain, the at least one preferably two non-conditional essential gene is selected from IscU operon, accC operon, glmM, ylaN, InfA, and dapA, and homologues thereof.

In one embodiment of the invention, when the production cell is a prokaryote, such as a Corynebacterium strain, the at least one preferably two non-conditional essential gene is selected from air, glmM, and homologues thereof In one embodiment of the invention, when the production cell is a eukaryote, the essential gene when non-conditional is selected from S. cerevisiae FOL1, MED7, RRP40, NOP8, PGI1, NEP1 and homologues thereof; and when conditional is selected from S. cerevisiae URA3, LEU2, TRP1, HIS3 and homologues thereof. In one embodiment of the invention, when the production cell is a filamentous fungi, such as Aspergillus, the essential gene is selected from ARG3, adeA, ERG10, PFS2 and TUB1 and homologues thereof.

In one embodiment of the invention, the at least one preferably two essential genes are conditionally essential genes leading to the synthesis of a product required for auxotrophic growth, or a protein product required for resistance to growth inhibitors such as an antibiotic, specific toxin, protoxin, or the like.

When the at least one preferably two essential genes are conditionally essential then the composition of the production cell's growth medium/growth conditions must be adjusted, such as using growth medium lacking specific nutrients or supplemented with growth inhibitors.

In one further embodiment, the production cell may comprise a combination of a nonconditional and a conditional essential gene. ^#1 An essential gene according to the invention is one encoding a protein that has at least 70%, 80%, 90%, 95% or even 100% amino acid homology with the protein encoded by the respective essential gene listed in the Table. ^#2 BioCyc/EcoCyc Database Collection is a large online collection of Pathway/Genome Databases – accessed through https://biocyc.org/ ^#3 SGD is the “Saccharomyces Genome Database” – accessed through https://yeastgenome.org/ ^#4 BL: accessed through https://www.genome.jp/kegg-bin/show_organism?org=bli ^#5 Aspergillusgenome.org The essentiality of a gene can be determined by creating cells in which the respective gene is knocked-out, where a non-conditional gene knockout will result in a loss of cell viability, or failure to grow, irrespective of growth conditions; while a conditional gene knockout will result in a failure to grow or loss of cell viability depending on the composition medium/conditions of cultivation. A suitable essential gene can be identified by Performing a test assay that swaps the native promoter of the assayed essential gene for an inducible promoter, such as the L- arabinose inducible p_BAD (in E. coli), xylose-inducible p_XYL (in Bacillus subtilis or Bacillus licheniformis) or galactose inducible pGAL (in S. cerevisiae or Pichia Pastoris), or plac (in E. coli or Lactobacillus) or pthiA (in Aspergillus). In such an essential gene assay, an integration DNA construct comprising the inducible promoter is chromosomally integrated into a host microbial cell using standard methods. In the case of prokaryotic organisms, the integration DNA preferably harbors an RBS catalog (e.g., Table 3) to direct translation of the essential gene at different rates to account for different baseline expression levels of different promoters and different baseline required expression levels required of essential genes to support growth at the corresponding wildtype specific growth rate. After targeted integration of the integration DNA, integrant cells are plated on plates supplemented with permissive conditions (temperature or concentration of inducers: >0.9 % L-arabinose, > 0.8% xylose, >1% IPTG, >2% galactose). Cells are subsequently tested for their synthetic addiction to the inducer condition in standard growth curve assays, and suitable essential genes can be identified as genes for which inducer-dependent growth rates can be identified. Inducer-dependent growth can be observed as a reduced growth rate of > 5 percent in absence of inducing conditions. Suitable essential genes are characterized by conferring less than < 5 % reduction in exponential-phase specific growth rate during optimal inducer conditions in production- relevant cultivation medium. Methods for identification of an essential gene are detailed in “Gene Essentiality – Method and Protocols” January 2015 [DOI 10.1007/978-1-4939-2398-4] which enables the skilled person to determine whether a gene is essential for microbial cell growth under specific growth conditions; including methods for mapping and identifying essential genes of Campylobacter jejuni; Streptococcus sanguinis; Porphyromonas gingivalis; Escherichia coli; Leptospirosis; Mycobacterium tuberculosis; Pseudomonas aeruginosa; and Candida albicans; primarily by screening transposon tagged libraries. Additionally, a variety of “computational tools” described therein enable the skilled person to directly predict and identify genes encoding essential proteins in a microbial genome, facilitated by the widespread availability of whole genome microbial sequences; and the structural features of the many known essential genes. Accordingly the skilled person is provided with both databases of essential genes; and various freely accessible on-line tools and algorithms for successfully and reproducibly identifying large numbers of essential genes in the genome of microbial cells without undue burden. It should further be noted that 82% of the essential genes in Saccharomyces cerevisiae encode essential proteins that are similar to a protein in another organism [Giaever G et al., 2002]; demonstrating that most essential genes, either known or identified in one micro- organism (e.g. yeast) will have a counterpart in other microorganisms – and could thus be identified in a microorganism of choice by a simple BLAST search. II.iii Burden-sensing promoter The microbial production cell of the invention comprises at least one preferably two burden-sensing promoter(s) operably linked to the at least one preferably two essential gene(s) encoding at least one preferably two protein(s), respectively, required for cell growth and/or survival, as described above. A burden-sensing promoter is one that is induced in a microbial production cell that is subject to a burden and/or fitness cost due to its production or over-production of a product. Typically, induction of the burden- sensing promoter will occur when a microbial cell’s production of a product results in a reduction in the cell’s growth rate that, in turn, is attributable to the fitness cost of production. Importantly, the at least one preferably two burden-sensing promoters, according to the invention, are induced by the “burden status” in the production cell resulting from production of a product, and not by the product per se. Since the use of the at least one preferably two burden-sensing promoter and essential gene to enhance productivity of a microbial production cell is largely independent of the product it is   engineered to make, the burden-addiction conferred by the invention supports a wide range of microbial production cell applications. Since the essential gene is operably linked to the burden-sensing promoter, its level of transcription and expression is only increased above a basal level when the burden- sensing promoter is induced. A suitable burden-sensing promoter is one that, when un- induced, permits a basal level of expression of the operably linked essential gene that is significantly limiting for, or prevents cell growth and/or survival. Basal level expression of essential gene is a level sufficient to support growth of a cell under production conditions (or during an exponential growth phase) at a level equal to or less than 10, 20, 50, 90 or 95% of the growth rate of a corresponding cell wherein said essential gene is operably linked to its native promoter. This basal level of expression of essential gene will be exerted in burden addicted cells when the burden-sensing promoters is not activated by the burden of efficient product synthesis, and thereby, the resulting lowered growth rate will constitute a selective disadvantage upon lack of product synthesis. Cells comprising burden-sensing promoter in the un-induced state, will grow significantly slower than cells where the essential gene is operably linked to its native promoter (example 1, figure 2). By contrast, microbial production cells comprising the burden- sensing promoter linked to an essential gene exhibit a significantly increased growth rate when the cells synthesize the desired product as compared to cells not producing the product (example 1, figure 3). Furthermore, microbial production cells of the invention, despite the inherent burden of producing a product, can surprisingly achieve an initial growth rate that is indistinguishable from the growth rate of parent microbial cells, devoid of gene(s) encoding the product or metabolic pathway for biosynthesis of the product, and from which the microbial production cell was derived (example 1, figure 4). Hence under production conditions, a microbial production cell of the invention will have a selective advantage over an escape variant of the production cell that ceases to synthesize the product, and thereby depress the escape rate. To increase production of desired products and further prolong the period during which the production cells are productive, more than one burden-sensing promoter may be suitable to use to promote growth of beneficial cell variants. By using two or more different burden-sensing promoters to addict a production cell, the highly productive cell variant can more uniquely be differentiated from less productive (genetic/non-genetic) cell variants that over time may arise from the same ancestral starting cell. For example, environmental factors e.g. resulting from outside stresses in a production culture may to some extent activate even less productive cells and thereby permit growth.   By using more than one burden-sensor to control essential genes, the transcriptional space that directs the imposed addiction-based selection regime can be further controlled. This may be important to limit potential cellular escape modes in which the burden addiction response of a single burden biosensor may permit sufficient growth to allow production declines. Table 2 provides a non-exhaustive list of potential burden-sensing promoters. In one embodiment, the burden-sensing promoter may be a native promoter, or a modified native promoter, with respect to the microbial production cell, while being heterologous with respect to the essential gene. In one embodiment, the promoter contains native TF/sigma factor binding sites. Suitable burden-sensing promoters are those of the E. coli σ32 regulon since they are generally upregulated in response to over-expression of proteins and their intracellular aggregation. A non-exhaustive list of E. coli σ32 promoters is included herein in Table 2, while additional suitable promoters are identified by Nonaka et al, 2006. In one embodiment, the burden-sensing promoter is a non-vegetative σ factor regulated promoter. In a preferred embodiment, the burden-sensing promoter is activated via the σ³² regulon, and can be selected from among promoters of the E. coli genes htpG, ibpA, clpB, yccV, grpE, ycjX, ldhA, mutM, ybbN, prlC, groES, fxsA, and htpX. In one embodiment, the burden-sensing promoter is the promoter of the mutM gene. mutM encodes a functionally conserved DNA-glycosylase responsible for initiating repair of one of the most common oxidative stress induced DNA damage, i.e. oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG) (Jain et al, 2007). Other suitable burden-sensing promoters may be selected from among promoters of the E. coli genes rrnB, rrnE, cspD, katE, xthA, uspE, gadB, ahpC, katG, grxA, oxyS, poxB, trxC. Burden-sensing promoters also include promoters related to a cell’s carbon nutrient status and growth phase. Accordingly, in one embodiment, the burden-sensing promoter is the promoter of the toxin gene, cspD, which is activated by reduced growth rates and carbon starvation (Uppal et al., 2014; Yamanaka and Inouye, 1997) and hence predicted to be differentially up-regulated in cells subject to burden or fitness cost due to production of metabolites.   In one embodiment, the burden-sensing promoter is a ribosomal RNA promoter, in particular a promoter of rrnB and rrnE genes, that respond to the nutritional supply, are induced by the Fis protein, and are inhibited by ppGpp alarmone. Rrn promoter activity is induced in cells with high protein production, as seen in their early exponential growth phase (Nonaka et al, 2006). In one embodiment, the burden-sensing promoter is one responsive to oxidative stress associated with metabolic burden and microbial heterologous production (Dragosits & Mattanovich, 2013). Oxidative stress response promoters include those promoters regulated by E. coli OxyR or its homologs such as Bacillus PerR, which activates several genes associated with amelioration and protection of the cell in response to oxidative damage; and promoters of genes belonging to rpoS (σ^S) that are upregulated during overexpression of proteins primarily during stationary growth phase. For Bacillus, suitable burden-sensing promoters may be selected e.g. among HrcA regulated promoters including the promoter of groES. Other suitable burden-sensing promoters may be selected from among promoters of the B. subtilis genes ctsR, dnaK, perR, hrcA, spx, sigB, yflT, mutM. For Corynebacterium, such suitable burden-sensing promoters may be selected from among katA, cplX, mutM and groES. For Saccharomyes, suitable burden-sensing promoters may be selected from among promoter of genes KAR2, PDI1, SAA1, FPR2, RPL3, RPL6A, RPL28, OGG1, RAD51, RAD54. For Aspergillus, such suitable burden-sensing promoters may be selected from among bipA, PDI, clxA and the homologues of S. cerevisiae OGG1, RAD51 and RAD54.         *** For Aspergillus, Aspergillusgenome.org is a database (AspGD) featuring the genomes of relevant Aspergillus species. The basal expression level and specific response curve upon induction/activation of the different burden-sensing promoters (e.g. in Table 2) vary, which can be exploited when selecting a burden-sensing promoter having the best match (in terms of strength) to a specific microbial production cell and its product. In some cases, the gene of interest is part of an operon, in which case the first gene of the operon is preferably used to define the relevant promoter sequence. Suitable burden-sensing promoters that are induced by the burden and/or fitness cost in a microbial production cell during production can be selected from public databases known to the skilled person, for example https://ecocyc.org for E. coli promoters; https://bsubcyc.org for Bacillus promoters (which also works for Corynebacterium promoters), their respective prokaryotic homologues; https://yeastgenome.org for Saccharomyces cerevisiae promoters, and their fungal homologues; and https://aspergillusgenome.org is a database (AspGD) featuring the genomes of relevant Aspergillus species. Suitable burden-sensing promoters can also be validated in a test assay in which a candidate burden-sensing promoter is operably linked to a gene encoding a fluorescent protein (e.g. green fluorescent protein) and integrated into a microbial production cell genetically engineered to synthesize a product, and integrated into a corresponding non- producing cell (control), which was isolated following a serial dilution cultivation experiment in production medium for between 50-150 cell generations or from the end of a large scale fermentation culture. Burden-sensing promoters induce transcription of the candidate burden-sensing promoter operably linked to fluorescent protein expression in producing cells compared to the non-producing cell (control) by at least 5 %, 7.5 %, 10 %, 15 %, 25 %, 60 %, 150 %, 300 % in the production cell compared to the activity in the isolated corresponding non-producing cell. II.iv Matching a burden-sensing promoter to a microbial production cell Microbial production cells, in response to the burden or fitness cost of producing a product, exhibit a transcriptional state characterized by the expression of certain genes, whose expression is not induced in a corresponding non-productive parent cell or a non- productive escape cell derived from a microbial production cell during production fermentation. The physiological nature of the burden or fitness cost will depend on the product synthesized by a given microbial production cell, and will be reflected in the   types of genes whose expression is induced. The identity of the gene promoters induced in a cell producing a given product can be determined by techniques well-known in the art (e.g. transcriptomics, see example 7). Since many of the induced gene promoters will be related to, or correspond to those listed in Table 2, these provide a starting point for finding a matching burden-sensing promoter for a given microbial production cell. The choice of promoters can be extended by identifying 5 to 15 promoters of genes found to be specifically upregulated in the production strain of interest. As illustrated in the examples herein, the method used to match a burden-sensing promoter operably linked to an essential gene to a microbial production cell in a product-specific manner, is experimentally fast and can be conducted in simple laboratory setups. II.v Translation control elements Regulating the growth of a microbial production cell of the invention by means of essential gene expression, requires that the response threshold and curve of the burden- sensing promoter and the expression level of the essential gene are balanced; such that basal level essential gene expression supports limited or no growth, while in highly productive cells the induced burden-sensing promoter drives sufficient essential gene expression to support a significantly increased growth rate, preferably a growth rate similar to that of productive cells lacking the burden-sensing promoter of the invention or ^5 % lower, when measured in the exponential growth phase. One suitable approach to balancing the burden-sensing promoter’s burden-response to the expression level of the essential gene is to modify the translational strength of the essential gene. In bacteria, translational strength is defined by the Shine- Dalgarno/ribosome binding site (RBS) sequence directly upstream of the start codon, while in eukaryotic cells translation initiation regions, translation initiation sites (TIS) or Kozak elements can be used to modify translational strength. RBSs in E. coli conferring a broad range of translational strengths are provided in Table 3.1, while further examples can be found in the literature (e.g. Bonde et al, 2016). A skilled person in the art can balance the translational strength of the regulated essential gene by constructing four variants of the ribosomal binding site for each burden-sensing promoter (Rugbjerg et al, 2018, PNAS), and testing which variant enables a selected essential gene to effectively regulate the growth rate of a cell. Exemplary RBSs for use in Bacillus licheniformis or Bacillus subtilis are provided in Table 3.2. Exemplary TISs for use in Pichia are provided in Table 3.3.   II.vi Engineering burden-sensing promoters and translational control element A native burden-sensing promoter of choice is generally encompassed by the -1 to -300 bp region (upstream) of the native regulated ORF in prokaryotic organisms and the -1 to -500 bp region in eukaryotic organisms. Core promoters that must be included and the sequence boundaries of their regulatory sites e.g. transcription factor binding sites, are common general knowledge, such as for σ32 (Nonaka et al, 2006). The translational control element/RBS can be added downstream of the promoter, to avoid alteration in the regulatory properties of the selected promoter sequence. II.vii Improved production levels A surprising advantage of the microbial production cell of the invention, in addition to having an increased initial growth rate as compared to non-productive cells, is that the cells retain significantly improved productivity during a large scale fermentation (simulated in example 2; figure 6), over many cell divisions, compared to microbial   production cells lacking engineered burden-addiction. The surprising advantages conferred by burden-addiction in a microbial production cell of the invention are obtained irrespective of the type of product synthesized – as illustrated by the range of products synthesizes by microbial production cells in examples 2-5. According to one embodiment, the microbial production cell of the invention is characterized by improved product yield following at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 100, 150, 250 or 400 generations of cell division from a single cell, compared to a non-burden-addicted production cell following the same generations of cell division. Product yield is measured as moles or grams of product produced per unit substrate. In one embodiment, production levels are increased by at least 10, 25, 50, or 80% following at least 50 generations of cell division from single cell, compared to a reference non-burden-addicted production cell following the same generations of cell division. Universal practice in the engineering of microbial cell factories has aimed at minimizing the burden known to arise due to maintenance and expression of heterologous pathways. The present invention provides a counter-intuitive solution to improving cell factory yields, since it makes microbial cell growth and survival dependent on the cell producing its product while being subject to a constant burden or fitness cost. While not wishing to be bound by theory, it is speculated that the selection pressure placed on a microbial production cell of the invention and its descendants, where only productive cells in a state of burden can grow/survive, may serve to progressively select for highly- burdened cell sub-populations during long-term culture in a starting population of initially isogenic cells. III. Methods for preparing and identifying cells of a burden-addicted microbial production strain In a preferred method of performing the invention, a burden-addicted microbial production strain is prepared and identified by the following steps. To obtain the best working relation between burden sensing promoter and essential gene, it may be relevant to prepare and test different candidate burden-sensing promoters with different RBSs for regulating a selected essential gene. III.i Identification of burden-sensing promoter candidate(s) in a specific production strain Candidate burden-sensing promoters listed in Table 2 may be tested. Alternatively, unique, positively differentiating gene transcripts detected in a specific production strain   of interest may be identified; whose respective promoters provide a source of candidate burden sensing promoters. Preferably such differentiating gene transcripts are identified by comparing the transcript profile of a microbial production strain (during production) with an isolated genetic or non-genetic escape mutant variant(s) derived from the corresponding production strain, where such escape mutant variant is characterized by at most a 50 % lower production rate. Positively differentiating gene transcripts can be identified by use of transcriptomics; for example by RNA sequencing of the transcribed RNA extracted from productive microbial production cell(s) compared to the non/low- producing escape mutant variant. Identified promoters can be tested in combination with a given essential gene in a microbial production cell. A method for identifying at least one preferably two burden-sensing promoters is illustrated in example 7; where after said promoter(s) is operably linked to an essential gene in a chosen microbial production cell for testing. III.ii Introduction of burden-sensing promoter in growth-regulating processes The at least one preferably two promoter(s) selected from the list of general candidate burden-sensing promoters (Table 2) or from identified specific burden-sensing promoter(s) (see IIIi), are tested by operably linking it to an essential gene in the microbial production cell. Different translation strengths of the essential gene can be simultaneously tested by providing alternative RBS sequences for the cognate essential gene; as well as testing different essential genes (as describe in section Iiii). The essential gene may be native or heterologous with respect to the microbial production cell. When the essential gene is a native gene, its cognate native promoter may be disrupted (i.e. made non-functional) by e.g. mutation or deletion events, and the burden-sensing promoter (that is heterologous with respect to the essential gene) is then operably linked to the native essential gene by targeted introduction. In another embodiment, the native essential gene promoter is replaced with the burden-sensing promoter by targeted introduction. Suitable methods for targeted introduction of a genetic sequence in microorganisms include recombineering in bacteria, e.g. Lambda Red recombineering in E. coli; while homologous recombination can be used in yeasts and filamentous fungi. Both concepts can optionally be used in combination with CRISPR to increase gene replacement efficiency.   III.iii Screening for balanced burden-sensing and growth-regulation Microbial production cell clones comprising an inserted burden-sensing promoter operatively linked to an essential gene are then screened to identify clones where the burden-sensing promoter regulates an essential gene in a growth-controlling manner. For example, the growth of a number of such clones (e.g.8-96 clones) is compared with cells of a corresponding non-burden-addicted microbial production strain, under conditions were product production is controlled (e.g. using microbial production strains where the production gene is inducible). When cell growth is measured under conditions where product synthesis by both strains is low/absent; a suitable burden-addicted production clone is one that exhibits a significant, and preferably at least 5 %, lower growth rate than the non-burden-addicted production strain (Example 1, Figure 2). If instead production is high/present; the up- regulation of essential gene expression in a suitable burden-addicted production clone, means that the difference in growth rate is significantly reduced or eliminated when compared to the corresponding non-burden addicted production strain. III.iv Validation of non-perturbed central and production metabolism Changes in transcriptional regulation or expression of essential genes can lead to unwanted, indirect perturbation of the production genes and central carbon or nitrogen metabolism, compromising product formation and in turn reduced burden in a burden- addicted microbial production cell. In order to exclude such clones, cell growth is measured under conditions where both strains synthesize product; where a suitable burden-addicted production clone is one that exhibits a growth rate equal to or lower than the non-burden-addicted production strain (as seen in example 2, figure 7). III.v Screening for growth-rate stability Clones that fulfil the above criteria are tested for growth rate stability under production- mimicking conditions by cultivation for at least 20 generations of cell division, e.g. by serial passage and measurement of growth rates, or by taking samples from various steps in a scaled-up production process. Suitable burden-addicted microbial production cells maintain growth rates lower than the non-burden-addicted production strain over time (e.g. example 2, figure 7).   IV. Method for producing a desired product using cells of a burden-addicted microbial production strain A second aspect of the present invention concerns a method for producing a desired product comprising the steps of: a. providing at least one microbial production cell genetically engineered to synthesize a product, wherein said at least one cell further comprises at least one preferably two essential gene(s) operably linked to at least one preferably two burden-sensing promoter(s), respectively, and wherein said promoter(s) are heterologous with respect to said essential gene, b. introducing the at least one genetically modified microbial cell into a cultivation medium comprising substrate for production of said product, c. recovering said product synthesizes by said culture, wherein synthesis of the product confers a burden and/or fitness cost on said at least one cell, and wherein expression of said at least one preferably two essential gene(s) are up-regulated relative to basal level expression of said at least one preferably two essential gene(s) when said at least one preferably two burden-sensing promoter(s) are induced by said burden and/or fitness cost; and wherein a lack of said product synthesis in said at least one production cell or progeny cell thereof reduces growth rate of said at least one cell. The method for producing the desired product may comprise the step of providing a cell culture of said at least one microbial production cell genetically engineered to synthesize a product; and introducing the cell culture of the at least one genetically modified microbial cell into a cultivation medium comprising substrate for production of said product. The cells are cultured in the cultivation medium to allow the cells to produce the desired product and to support growth and multiplication of the cells. The time of cultivation may be optimized depending on that the desired product is. The method for producing the desired product may further comprise a step of isolating the product and/or formulating of the product into a composition, such as a nutritional, pharmaceutical, cosmetic, detergent, lubricant, or fuel composition   V. Use of cells of a burden-addicted microbial production strain for producing a desired product A third aspect of the present invention concerns the use of a burden-addicted microbial production cell of the invention for producing a desired product, wherein a lack of product synthesis in said a burden-addicted production strain or progeny cell thereof reduces growth rate of said strain even if product is present in the intracellular or extracellular environment. EXAMPLES Example 1: Engineering burden-addicted E. coli strains producing recombinant human growth hormone 1.1 Maintenance and expression of heterologous production genes by a cell factory constitutes an unnatural burden on its cells The growth rate of parent E. coli BL21(DE3) cells was compared with an engineered derivative transformed with the plasmid, pEG34, comprising a gene encoding recombinant human growth hormone (hGH) fused to Green Fluorescent Protein (GFP). 1.1.1 Materials and Methods Cells of the host strain E. coli BL21(DE3) (E. coli Genetic Stock Center at Yale University), were made electrocompetent and transformed by standard electroporation methods (1800 V, 25 µF, 200 Ohms, 1 mm cuvette width) with the plasmid pEG34, and plated on LB agar plates containing chloramphenicol.   *camR gene [SEQ ID No: 103] encoding chloramphenicol resistance protein [SEQ ID No: 104] is in pEG34 [SEQ ID No: 101] located at nucleotide positions 3768-4427 on the complementary strand; is in pEG0 [SEQ ID No: 105] located at nucleotide positions 2339-2995 on the complementary strand; is in pENDU5CAM [SEQ ID No: 106] located at nucleotide positions 1617-2276 on the complementary strand; and is in pMevT [SEQ ID No: 108] located at nucleotide positions 6585- 7244 on the complementary strand. Pre-cultures of single colony transformants were cultured in 96-well microtiter plates on 200 µL 2xYT medium (16 g/L tryptone, 5 g/L NaCl, 10 g/L yeast extract) containing 500 µM IPTG and 30 mg/L chloramphenicol at 37 °C with fast horizontal shaking in an Elx808 plate reader (Biotek) with reads at OD630 every ten minutes. OD630 values were background-subtracted using the OD630 value of the first read. 1.1.2 Results E. coli cells comprising the hGH-GFP expression plasmid, pEG34, grow slower than cells of the parent E. coli host from which is was derived (figure 1); illustrating the burden or load placed on cells as a result of allocating cellular resources to maintenance and expression of a synthetic construct. 1.2 Engineering a burden-addicted cell factory Burden-addiction strains of the parent E. coli host were genetically engineered to incorporate a genetic circuit designed to confer a selective fitness advantage on productive cells in a cell factory. Specifically, a burden-sensing promoter (p_mutM, p_yccV or pycjX), was substituted for the native promoter of the essential gene operon folP-glmM in the E. coli BL21(DE3) chromosome. The genetic circuit further included an RBS, between the promoter and essential gene, where four different RBSs (Table 3) were tested for modulating the expression level of the essential gene. The growth rate of strains comprising the genetic circuit was compared with the parent host strain E. coli BL21(DE3). 1.2.1 Materials and Methods Burden-sensing promoters: The promoters, pmutM, pyccV or pycjX, were generated by PCR amplifying a 0.3 kb region immediately upstream of the respective gene (see Table 3) using primers specified in Table 5 and genomic DNA derived from lysed E. coli BL21(DE3)   cells as template. The PCR mix: 10 µl MQ water, 2 µl forward primer (10µM), 2 µl revers primer (10µM), 1µl DNA template, 15 µl Phusion U MasterMix (Thermo Scientific). The PCR reaction protocol: 95 ^C for 180 sec (1x); 95 ^C for 20 sec, 68-58 ^C (touchdown) for 30 sec, 72 ^C for 60 sec (35x); 72 ^C for 300 sec (1x); leave at 15 ^C. USER cloning was used to generate integration sequences comprising the amplified promoter region fused to a linear 1.5 kb DNA fragment containing a kanR gene for selection of correct recombineering products and a 221 bp targeting sequence identical to 221 bp directly upstream of the folP gene. The amplified promoter region and kanR- folP fragment were fused by mixing them in equimolar amounts and adding 1µl 10x T4 ligation buffer (Thermo Scientific) and 0.75 µl USER enzyme (New England Biolabs), in a total 10µl reaction volume. The USER reaction was placed at 37°C for 30 minutes. The reaction was then placed at room temperature for 15 minutes followed by the addition of 0.75 µl T4 DNA ligase (Thermo Scientific) and incubation at room temperature for 30 minutes. An example of such integration sequence comprising promoter, KanR resistance gene, and folP targeting sequence can be found in the sequence listing SEQ ID NO.140 (s9_pmutM_folP). The ligated product was then amplified using primers ‘rev’   (according to specific promoter in Table 5) and P493 (Table 5) to approximately 250 ng/µl using the PCR reaction protocol: 98 ^C for 180 sec (1x); 85 ^C for 20 sec, 72-68 ^C (touchdown) for 30 sec, 72 ^C for 60 sec (35x); 72 ^C for 5 min (1x); leave at 15 ^C. Primer overhang provided 50 bp folP identical targeting sequence to direct recombineering to the folP locus. Chromosomal integration of burden-addiction promoters: promoters were integrated upstream of the folP gene in the genome of E. coli BL21(DE3) cells as follows: 100 ml of 2xYT medium containing tetracycline were inoculated with 600µl of BL21(DE3) overnight culture pre-transformed with pSIM5-tet (Koskiniemi et al, 2011). The cells were cultured at 30°C, and upon reaching OD600=0.20, the culture was transferred to a 42°C shaking bath for 15 minutes to allow for the expression of the recombineering enzymes located on pSIM5-tet. The culture was then transferred to 2x cold 50ml centrifuge tubes; centrifuged at 4000g for 10 min; the supernatant was discarded; and the remaining cell pellets were washed with 20 ml ice-cold 10% glycerol. The partially re-suspended cells were then centrifuged at 4000g for 6 min; the supernatant was discarded; and the cell pellets washed with 20 ml ice-cold 10% glycerol. The partially re-suspended cells were centrifuged at 4000g for 6 min; each cell pellet was carefully re-suspended in 495 µl ice-cold 10% glycerol and pooled. 90 µl of re-suspended cells were added to electroporation cuvettes each containing 1 µl (>250 ng) of one of the burden-sensing promoter integration sequences. The cells were electroporated with the following settings: 1800 V, 25 µF, 200 Ohms, 1 mm cuvette width. 900 ml 2xYT-media was added to the cuvette straight after electroporation and the cells were left to recover for 1.5 hour at 37°C in 1.5 ml Eppendorf tubes. The electroporated cells were incubated at room temperature overnight to allow time for recombination; and subsequently plated on LB agar plates containing 50 mg/L kanamycin and cultured at 37°C overnight to ensure curing for pSIM5-tet. Correct targeting to the essential gene locus was validated using primers P525 and P526. Generally, single kan^R colonies were picked having a size average or smaller than the population of colonies. Burden-addiction promoter integration in the genome of the kanR selected colonies was validated by colony PCR using Taq DNA polymerase and primers targeted to the folP promoter region. Additionally, the identity of the RBS in the selected colonies was determined by Sanger sequencing. Subsequently, the growth rate of strains comprising the genetic circuit was compared with the parent host strain E. coli BL21(DE3) by cultivation in 200µl 2xYT supplemented with 500 ^M IPTG at 37 ^oC with horizontal shaking.   1.2.2 Results The growth rate of the selected burden-addicted E. coli strains were slower, to various degrees, compared to parent E. coli host from which they were derived (figure 2; showing growth of burden-addicted strains s5.0#3, s7.0#8, and s9.0#8 wherein essential genes folP-glmM are controlled by burden-sensing promoters p_yccV, p_ycjX, and pmutM, respectively). Since cellular growth is dependent on the level of expression of the essential folP-glmM genes, it may be concluded that the level of essential gene expression driven by the respective burden-sensing promoter in a non-productive, burden-addicted strain is insufficient to support growth at the level of a wildtype parent strain. The observed reduction in growth rate in these burden-addicted strains provides a measure of the penalty that may be exerted on a non-producing cell that evolves spontaneously during cultivation of a cell factory population comprising the burden- addiction genetic circuit. The degree of penalty is determined by the choice of the burden-sensing promoter combined with the strength of the chosen RBS. The larger the penalty – the wider the window for “negative selection” of any low- or non-producing variants (e.g. resulting from mutation in production genes) spontaneously arising during cultivation. 1.3 Production increases growth rate of a burden-addicted cell factory The burden-addicted E. coli strains, which conferred a growth penalty for non-production (figure 2), were used as host strains for demonstrating the selective fitness advantage for burden-addicted cells that are productive. 1.3.1 Materials and methods The slowest-growing clones of the generated burden-addicted E. coli strains (s9.0#8; s5.0#3; and s7.0#8) were made electrocompetent and transformed by standard electroporation methods (1800 V, 25 µF, 200 Ohms, 1 mm cuvette width) with respectively pEG34 or pEG0 (Table 4), and plated on LB agar plates containing chloramphenicol and kanamycin. Single colony transformants were cultured over-night on 2xYT medium containing chloramphenicol, and then used to inoculate 96-well microtiter plates comprising 200 µL 2xYT medium (500 µM IPTG, chloramphenicol) and cultured at 37 °C with fast horizontal shaking in an Elx808 plate reader (Biotek) with reads at OD630 every ten minutes. OD630 values were background-subtracted using the OD630 value of the first read.   E. coli strains were each transformed with the plasmid pEG34 or pEG0 (Table 4), and their growth rate properties were measured when the cells were induced to synthesize the recombinant protein, hGH fused to GFP. 1.3.2 Results The burden-addicted strain, E. coli strain s9.0#8, comprising the burden sensing promoter pmutM controlling of the folP-glmM essential genes expression, exhibited the slowest growth of the strains tested when compared to the parent E. coli BL21 (DE3) strain (figure 2). This reduction of exponential growth rate is reversed in cells of the E. coli strain s9.0#8 transformed with the pEG34 plasmid and induced to express the recombinant protein, hGH-GFP, in contrast to cells of E. coli strain s9.0#8 transformed with the empty plasmid, BEG0 (figure 3). This demonstrates that the burden-sensing promoter pmutM in E. coli strain s9.0#8 is induced by the burden or load on the cells brought about by synthesis of the recombinant protein hGH-GFP. This, in turn, leads to up-regulated expression of the folP-glmM genes and enhanced exponential growth. The synthesis of hGH-GFP in each of the burden-addicted strains, E. coli strain s5.0#3, s7.0#8, and s9.0#8 harboring the pEG34 plasmid, not only led to a reversal of the reduction of exponential growth rate; but additionally the growth rate of these strains was indistinguishable from growth of the parent E. coli BL21 (DE3) (figure 4). In summary, this example illustrates that the burden-addiction genetic circuit can be used to confer a selective fitness advantage on those cells of a cell factory whose synthesis of proteins or metabolites is sufficiently high to constitute a burden or load; and that this burden or load is detectable by a burden-sensing promoter operably linked to an essential gene in the cells. By contrast, non-productive variant cells (e.g. resulting from production gene mutations), that spontaneously appear during cultivation of the burden-addicted cell factory, will be subject to a negative selection pressure, since their growth will be slowed to a rate supported by the basal expression of the essential gene. A reduced growth rate is, of itself, sufficient to slow the increase in frequency of such variant cells in the cell factory and thereby delay the decline in cell factory productivity over time. Example 2: Burden addicted E. coli strains producing human growth hormone show enhanced long-term production stability Burden-sensing promoters are shown to be promoters that can sense and be activated by a burden-induced state in a cell resulting from the cells synthesis of a recombinant protein, hGH-GFP. Once activated, burden-sensing promoters are shown to elevate   expression of an essential gene, folP-glmM, to a level sufficient to confer a selective growth advantage on a cell when compared to a non-productive cell. In order to maximize the dynamic range of essential gene expression in response to its cognate burden sensing promoter, the burden-sensing promoter is randomly combined with variant RBS coding sequences (Table 3) conferring different translational strengths. Promoters having burden-sensing properties suitable for use in a burden-addiction genetic circuit are shown to include heat-shock-, DNA damage-, oxidative stress response-promoters and rRNA promoters, as illustrated by the following engineered production strains cultured under simulated large-scale production conditions. Firstly, a number of clones harboring random variant RBS coding sequences were selected for each of the burden-sensing promoters, and their hGH-GFP synthesis was followed over many cell divisions in order to demonstrate their relative ability to elevate/preserve recombinant hGH-GFP synthesis over time. 2.1 Materials and Methods Burden-sensing promoters: The promoters, pyccV, pycjX, pibpA, pgrpE, pldhA and pybbN, and their respective integration sequences were generated by PCR and USER cloning as described for Example 1.2.1, using the primers specified in Table 6; while the ligated products were amplified using primers ‘rev’ (according to specific promoter in Table 6) and P493 (Table 5).   Chromosomal integration of burden-addiction promoters: each of the promoters was integrated upstream of the folP gene in the genome of E. coli strain BEG34 (corresponding to E. coli BL21(DE3) harboring plasmid pEG34), as follows. Cells of the E. coli strain BEG34, pre-transformed with the recombineering plasmid pSIM5-tet, were prepared and transformed by electroporation with each of the promoter integration sequences, as described in example 1.2.1. Following the described steps of recombineering and curing of pSIM5-tet, eight colonies were picked from each plate, corresponding to eight clones with the same promoter integration but having a random variant RBS sequence (see Table 6). Each clone was then transferred to a well of a 96- well plate containing 200 µl 2xYT supplemented with chloramphenicol for maintenance of the plasmid, pEG34. 2 µl of each cultured clone was used to validate promoter   integration via colony PCR as described in example 1.2.1; prior to freezing the 96-well plate. Short term hGH-GFP production screening assay: The frozen 96-well plate was thawed and a pin replicator used to transfer cells into a new 96-well plate containing 200 µL 2xYT supplemented with chloramphenicol. This plate was sealed with Breathe-Easy sealing membrane (Sigma-Aldrich) and placed in a SynergyH1 plate reader (Biotek) overnight at 37°C and 754 rpm linear shaking for 20 hours; and the OD (600nm) and GFP fluorescence (ex/em 485nm/528nm) of each well was measured every 10 minutes over a 20 hour period. The plate was then placed in a regular tabletop plate shaker at room temperature for 4 hours followed by the transfer of 2 µL culture from each well to a new 96-well plate containing 200 µL 2xYT supplemented with chloramphenicol and 0.5mM IPTG. The new 96-well plate was likewise sealed with Breathe-Easy sealing membrane (Sigma-Aldrich) and placed in a SyngergyH1 plate reader overnight at 37°C and 754 rpm linear shaking for 20 hours; and the OD and GFP fluorescence of each well was measured at 600nm and ex/em 485nm/528nm every 10 minutes for 20 hours. The following day 2 µL culture was transferred to a new 96-well plate with 200 µl 2xYT supplemented with chloramphenicol and 0.5mM IPTG and the process was repeated every day for 6 days in total. Long term hGH-GFP production assay (figure 6): A single selected colony from each burden-addicted hGH-GFP producing strain was used to inoculate a 15 mL Greiner culture tube containing 4 mL 2xYT supplemented with chloramphenicol and 0.5mM IPTG. The cultures were grown for 23 hours at 37°C on a 250 rpm shaking table. 2 µL of each culture were then seeded in 2000x dilution (corresponding to approx. 11 generations of cell division) into a new Greiner culture tube under identical conditions, where these method steps were repeated for a total of 12 seeds. After each passage, 200 uL samples of the culture were taken to determine hGH-GFP synthesis by measuring OD600 and GFP fluorescence (ex/em 485nm/528nm) in a SynergyH1 plate reader (Biotek). Long term hGH-GFP production assay (figure 8): A single selected colony from each burden-addicted hGH-GFP producing strain was used to inoculate a 24 deep-well plate carrying 1.8 mL 2xYT supplemented with chloramphenicol and 0.5mM IPTG. The cultures were grown for 23 hours at 30°C on a 200 rpm shaking table. 2 µL of each culture were then seeded in 1000x dilution (corresponding to approx. 10 generations of cell division) into a new deep well plate under identical conditions, where these method steps were repeated for a total of 9 seeds. After each passage, 200 uL samples of the culture were taken to determine hGH-GFP synthesis by measuring OD600 and GFP fluorescence (ex/em 485nm/528nm) in a SynergyH1 plate reader (Biotek).   Growth rate measurements of chosen strains: Selected high-hGH producing strains, s3.6#2, s6.6#6, s7.6#8 and s10.6#7, were streaked on LB agar plates containing chloramphenicol and kanamycin. BEG34 and BEG0 strains were streaked on LB agar plates containing chloramphenicol. 7 colonies from each plate were used to inoculate a 96-well plate containing 200 µl 2xYT supplemented with chloramphenicol; which were sealed with Breathe-Easy sealing membrane (Sigma-Aldrich). Their growth was measured in a SynergyH1 plate reader (Biotek) overnight at 37°C and 754 rpm linear shaking. 2 µl samples from each well were transferred to wells in a new 96-well plate containing 200 µl 2xYT supplemented with chloramphenicol and 0.5mM IPTG. The 96- well plate was sealed with breathe-easy film and incubated for 20 hours at 37°C and 754 rpm linear shaking and growth (OD600nm) was measured every 10 minutes using a Synergy H1 plate reader. Strain catalogue: BEG34 = E. coli BL21(DE3) carrying pEG34; BEG0 = E. coli BL21(DE3) carrying empty plasmid pEG0 sX.Z#Y = E. coli BL21(DE3) carrying pEG34, having wt folP promoter swapped for burden addiction-promoter X, wherein X refers to the promoter IDs in Table 2. The number Y following # indicates the selected clone from the pool of four RBS variants introduced by degenerate primer (Table 7). Z refers to the production genes for hGH- GFP (0 or 6), mevalonic acid (1), lysostaphin (4), respectively.   2.2 Results 2.2.1 Short term hGH-GFP productivity screening The productivity of four hGH-GFP producing strains comprising burden-sensing promoters selected from a group of heat shock promoters (pibpA, pgrpE, pycjX, and pybbN) and having one of four RBS coding sequence variants was tested under simulated large- scale production conditions, as follows. The strains were serially passaged by 100x back- dilution every day for 6 consecutive days corresponding to ca. 6 generation per seed. By day 6 (seed 6), several strains showed an elevated hGH-GFP synthesis compared to the non-burden addicted production strain BEG34, both during seed 1 and seed 6. The strains having high short term hGH-GFP productivity relative to strain BEG34 are s3.6#2 (pibpA), s6.6#6 (pgrpE), s7.6#8 (pycjX), and s10.6#7 (pybbN) – see figure 5. 2.2.2 Enhanced long term hGH-GFP productivity hGH-GFP productivity of burden-addicted strains, s3.6#2, s6.6#6, s7.6#8, s10.6#7, and non-burden addicted strains BEG34 and non-producing strain BEG0 was monitored under simulated large-scale fermentation by serial transfer. Following seed 2, burden- addicted strains s3.6#2, s6.6#6, s7.6#8, s10.6#7 and the non-burden addicted control strain BEG34 performed equally well in terms of hGH-GFP synthesis (figure 6. However, after an additional ca. 20 generations (seed 4), hGH-GFP productivity by the control strain, BEG34, had declined considerably; and by seed 6, the strain essentially ceased production. Surprisingly, the burden-addicted strain s7.6#8 (with pycjX controlling folP- glmM) still retained a productivity of around 50 % of seed 2 level. Additionally, burden- addicted strains s3.6#2 (with pibpA controlling folP-glmM), s10.6#7 (with pybbN controlling folP-glmM), and s6.6#6 (with pgrpE controlling folP-glmM) also showed significantly better long-term productivity as compared to the non-burden addicted strain BEG34. 2.2.3 Growth rate of burden addicted strains The growth rates of hGH-GFP producing burden-addicted strains s3.6#2, s6.6#6, s7.6#8 and s10.6#7 is no higher than the non-burden addicted hGH-GFP production strain BEG34; and hence their improved hGH-GFP production levels over time does not to simply stem from a reduced in initial production level and thus inherently lower burden, (figure 7). 2.2.4 Dependence of burden addiction genetic circuit on selected essential gene A hGH-GFP producing burden-addicted strain (s13.6#2 (fxsA)) comprising the heat shock promoter pfxsA controlling the essential gene folP-glmM was compared with a mutant derivative comprising a frame-shift folP abolishing folP expression, strain   s13.6#2evo (fxsA), whose growth was solely dependent on glmM expression. Although the loss of folP expression led to a lower growth rate (data not shown) in complex 2xYT medium, the essential gene glmM, alone is shown to be sufficient to confer enhanced long-term hGH-GFP production stability in the burden-addicted strain s13.6#2evo(fxsA) at levels comparable to the strain s7.6#8 (pycjX), when compared to the non-burden addicted hGH-GFP producing strain BEG34 (figure 8). 2.2.5 Use of ribosomal RNA promoters in the burden addiction genetic circuit In addition to heat-shock promoters, ribosomal RNA promoters such as p_rrnB and p_rrnE are shown to be capable of sensing a burden-addicted state in a cell and, in response, to control essential gene expression such as to enhance long-term productivity in a hGH- GFP production strain. As seen in figure 9, the long-term stability of hGH-GFP production in strains s15.6.7 (prrnB) and s16.6.6 (prrnE), was significantly improved when compared to a non-burden addicted hGH-GFP producing E. coli strain BEG34, under simulated large scale production corresponding to around 110 generations of cell division. 2.2.6 Use of oxidative stress sensing promoters in the burden addiction genetic circuit Oxidative stress-sensing promoters such as p_poxB are shown to be capable of sensing a burden-addicted state in a cell and, in response, to control essential gene expression such as to enhance long-term productivity in a hGH-GFP production strain. As seen in figure 10, both production levels and long-term stability of hGH-GFP production in strain s29.6#3 (ppoxB), was significantly increased when compared to a non-burden addicted hGH-GFP producing E. coli strain BEG34, under simulated large scale production of seven serial passages corresponding to approximately 90 generations and time-extended cultivation for the final seed. As seen in figure 10, production using the burden addiction genetic circuit based on p_poxB surprisingly resulted in elevated production over time, indicative of the enrichment of non-genetically high performing variants in the population. In summary, this example illustrates that burden addiction enhances long-term stability and production in E. coli strains engineered to synthesize human growth hormone fused to GFP by coupling transcription of essential genes folP-glmM to any one of a E. coli heat shock, oxidative stress and DNA damage responsive promoter as well as an rRNA promoter.   Example 3: Burden addicted E. coli strains producing lysostaphin show enhanced long-term production stability Lysostaphin is a 27 kDa endopeptidase that cleaves crosslinking pentaglycine bridges in the cell wall peptidoglycan of Staphylococcus aureus resulting in cell lysis. This antibacterial agent can be synthesized recombinantly in E. coli. The burden-addiction genetic circuit comprising a promoter having burden-sensing properties is shown to enhance the production stability of such E. coli strains engineered to synthesize lysostaphin, as illustrated by the following burden-addicted E. coli strains producing lysostaphin under simulated large-scale production conditions. The productivity of the burden-addiction production strains is further shown to be optimized by selecting a strain where the burden-sensing promoter is combined with an RBS sequence (Table 6) conferring an optimized translational strength of the cognate essential gene. These advantages are exemplified by burden-addicted strain s6.4#5 (pgrpE), described below, that is shown to be more resilient to loss of production due to addiction to the burden or fitness cost resulting from lysostaphin expression. 3.1 Materials and Methods Burden-sensing promoters: A promoter integration sequence comprising the promoter pgrpE was generated as described for example 1.2.1, by amplifying the pgrpE promoter by PCR, using the p_grpE-specific primers specified in Table 6. Chromosomal integration of burden-addiction promoters: the grpE promoter was integrated upstream of the folP-glmM gene in the genome of E. coli strain BENDU5cam (corresponding to E. coli BL21(DE3) harboring a lysostaphin producing plasmid pENDU5cam), as follows. Cells of the E. coli strain BENDU5cam, pre-transformed with the recombineering plasmid pSIM5-tet, was prepared and transformed by electroporation with the pgrpE promoter integration sequence, as described in example 1.2.1. Following the described steps of recombineering and curing of pSIM5-tet, 5 colonies were picked, corresponding to clones with the same promoter integration but having a random variant RBS sequence (see Table 6). Each clone was then transferred to a 15 mL Greiner culture tube containing 4 mL 2xYT supplemented with chloramphenicol for maintenance of the plasmid, pENDU5cam, and cultured.2 µl of each cultured clone was used to validate promotor integration as described in example 1.2.1; prior to freezing. Lysostaphin production screening: The E. coli strain s6.4#5(p_grpE) having the highest lysostaphin-productivity was identified using a screening assay in which the three other potential combinations of RBS and pgrpE were cultured in 2xYT supplemented with   chloramphenicol in 96-well format over six serial passages of 1000x dilution (corresponding to 60 generations) as compared to the non-burden addicted production strain BENDU5cam. The strains were grown in 3mL 2xYT media containing 30 mg/L chloramphenicol in 15 mL culture tubes at 37°C for 21 hours. 150 µL of the cultures were mixed with 50 µl 50% glycerol and stored in a 96-well plate at -80°C. Additionally, 30 µL of the cultures were transferred to 3 ml fresh 2xYT media supplemented with chloramphenicol and grown for another 21 hours at identical conditions; and repeated for a total of 5 transfers where freeze-stocks were made of the overnight cultures with each transfer. Lysostaphin expression and detection assay: The 96-well plate containing all freeze- stocks from the 5 transfers were thawed on ice. 20 µL from each well were transferred to a 96-well plate containing 180 µL 2xYT supplemented with chloramphenicol, and cultured grown at 37°C in a plate reader until most wells had reached OD630=0.35; and then 10 µl 2xYT supplemented 30 mg/L chloramphenicol and 20 mM IPTG was added to each well to a final concentration of 1 mM IPTG, sufficient to induce lysostaphin gene expression. The induced cultures were grown at 37°C for 4 hours, and the plate was then centrifuged at 4000 RPM for 10 minutes. 100 µl supernatant was transferred to a new 96-well plate containing 100 µl overnight S. aureus culture. The S. aureus lysis in each well was monitored at OD630 on a plate reader at 37°C for 2 hours every 10 minutes. The S. aureus lysis rate was quantified by dividing the change in S. aureus OD by the timeframe of 60 minutes that the change occurred in. The equation can be seen below: The specific lysostaphin synthesis rate was determined by normalizing the measured rate to the final OD630 measurement of the respective production E. coli culture. 3.2 Results The burden-addicted lysostaphin-producing strains having a p_grpE promoter and one of four RBS coding sequence variants controlling expression of the essential genes, folP- glmM, were cultivated under simulated large-scale production conditions, achieved by serial passaging. As seen in figure 11, strain s6.4#5 (p_grpE) maintained significantly higher levels of lysostaphin production as compared to the steep reduction in production seen in the non-burden addicted lysostaphin-production BENDU5cam strain. Since the initial lysostaphin production rates for s6.4#5 (pgrpE) and BENDU5cam strains at seed 0   were the same; the smaller decline in production rates for s6.4#5 (p_grpE) is not due to an inherently lower initial burden from lysostaphin production. In summary: Burden-addiction was demonstrated to enhance long-term stability and production in E. coli engineered to synthesize secreted lysostaphin by coupling essential genes folP-glmM transcription to the E. coli heat shock promoter pgrpE. Example 4: Burden addicted E. coli strains producing mevalonic acid show enhanced long-term production stability E. coli BL21(DE3) cells were engineered to synthesize mevalonic acid by introducing a plasmid pMevT expressing a heterologous three-step enzymatic pathway (Martin et al., 2003) that converts glucose to mevalonic acid via the acetyl-CoA pool. A burden- addiction genetic circuit comprising a promoter having burden-sensing properties and controlling transcription of an essential gene is shown to enhance the production stability of such mevalonic acid-producing E. coli strains under simulated large-scale production conditions. The productivity of the burden-addiction production strains is shown to be further optimized by selecting a strain where the burden-sensing promoter in combined with a RBS coding sequence (Table 5) conferring an optimized translational strength of the cognate essential gene. Specifically, mevalonic acid-production strains comprising the burden-addiction promoter, pcspD ( an oxidative stress and glucose starvation sensing promoter), or p_mutM (heat shock/DNA damage-sensing promoter) controlling folP-glmM transcription, are both shown to be more resilient to loss of production than the non- burden addicted strain due to their addiction to transcriptional signals resulting from mevalonic acid production. 4.1 Materials and Methods Burden-sensing promoters: Promoter integration sequences comprising the promoters pmutM and pcspD were generated as described for example 1.2.1, by amplifying the pmutM and p_cspD promoters by PCR, using the p_mutM- and p_cspD-specific primers specified in Table 8.   Chromosomal integration of burden-addiction promoters: the p_mutM and p_cspD were individually integrated upstream of the folP-glmM gene in the genome of cells of the E. coli strain BL21(DE3)pMevT, harboring a lysostaphin producing plasmid pMevT, as follows. Cells of the E. coli strain BL21(DE3)pMevT, pre-transformed with the recombineering plasmid pSIM5-tet, were prepared and transformed by electroporation with each of the promoter integration sequences, as described in example 1.2.1. Following the described steps of recombineering and curing of pSIM5-tet, 5 colonies were picked, corresponding to clones with the same promoter integration but having a random variant RBS sequence (see Table 8). Each clone was then transferred to a well of a 96-well plate containing 200 µl 2xYT supplemented with chloramphenicol for maintenance of the plasmid, pMevT, and cultured. 2 µl of each cultured clone was used to validate promotor integration as described in example 1.2.1; prior to freezing the 96- well plate. Mevalonic acid production screening: The E. coli strains s19.1.1(p_cspD) and s9.1.4(p_mutM) having the highest mevalonic acid productivity were identified using a screening assay in which strains comprising the four other potential combinations of RBS and promoter were cultured in 96-well plates over six serial passages of 1000x dilution (corresponding to 60 generations) in parallel with the non-burden addicted production E. coli strain BL21(DE3)pMevT, as follows. The strains were grown in 200 μL 2xYT media containing 30 mg/L chloramphenicol and 0.5mM IPTG in microtiter plate sealed with breathe-easy seal at 37°C for 21 hours with horizontal shaking. 150 µL of the cultures were mixed with 50 µl 50% glycerol and stored in a 96-well plate at -80°C. Additionally, 2 µl of a 10-fold diluted culture was transferred to 200 μL fresh 2xYT media supplemented with chloramphenicol and 0.5mM IPTG and grown for another 21 hours under identical conditions. In total, 5 transfers identical to the one described above were made and freeze-stocks were made of the overnight cultures with each transfer.   Mevalonic acid synthesis and detection assay: The 96-well plate containing freeze-stocks from the second, fifth and sixth transfers were thawed on ice and used to inoculate 10 mL 2xYT with 0.5 mM IPTG and 30 mg/L chloramphenicol and cultivated at 37°C with horizontal shaking (250 rpm) for 54 hours. 300^μL aliquots from each culture were treated with 23^μL 20% sulfuric acid; vigorously shaken and then spun down at 13^000^×^g for 2^min. Supernatant (medium) samples were injected into an Ultimate 3000 high-performance liquid chromatography running a 5^mM sulfuric acid mobile phase (0.6^mL/min) on an Aminex HPX-87H ion exclusion column (300^mm^×^7.8^mm, Bio-Rad Laboratories) at 50^°C. A refractive index detector was used for detection. A standard curve for mevalonic acid was generated with mevalonolactone (Sigma-Aldrich) dissolved in 2xYT medium supernatant of a non-producing E. coli strain incubated under the same conditions. 4.2 Results The burden-addicted mevalonic acid-producing strains having the p_cspD or p_mutM promoters and one of four RBS coding sequence variants controlling expression of the essential genes, folP-glmM, were cultivated under simulated large-scale production conditions, achieved by serial passaging. As seen in figure 12, both selected burden- addicted strains s19.1.1(p_cspD) and s9.1.4(p_mutM) retained significantly higher levels of mevalonic acid production as compared to the steep reduction in production seen in the non-burden addicted mevalonic acid-production strain BL21(DE3)pMevT. Since the initial mevalonic acid production rates for each burden-addicted strain was the same and the control BL21(DE3)pMevT strain at seed 0; the smaller decline in production rates for strains s19.1.1(p_cspD) and s9.1.4(p_mutM) is not due to an inherently lower initial burden from mevalonic acid production. In summary: Burden-addiction is demonstrated to enhance long-term stability and production in E. coli engineered to synthesize mevalonic acid by coupling essential genes folP-glmM transcription to the E. coli oxidative stress and glucose starvation sensing promoter p_cspD, and the heat shock/DNA damage-sensing promoters p_mutM. Example 5: Evaluation of promoters as burden sensors to render yeast cells addicted to the burden of engineered recombinant protein production Promoters capable of sensing the burden or load on a cell brought about by recombinant expression of a protein or a biosynthetic pathway, and that then induce the expression of an essential gene, can be used to create a burden addiction genetic circuit tailored for use in yeast. A method for evaluating candidate promoters is illustrated in yeast cells   genetically engineered to synthesize recombinant human serum albumin (hSA) or insulin precursor (IP), optionally translationally fused to green fluorescent protein (GFP). To render growth responsive to the activity of a candidate burden-sensing promoter, the native promoter of an essential gene in the yeast Saccharomyces cerevisiae is genetically replaced with a candidate promoter using homologous recombination of linear DNA constructs transformed into the yeast cells using a standard selectable marker. By way of example, candidate promoters may be selected from upregulated promoters of ribosomal RNA genes, such as those transcribed by RNA polymerase I (Laferté et al., 2006), DNA damage sensing promoters (e.g. pOGG1), and unfolded protein response (UPR) promoters upregulated by the HAC1 transcription factor (Kimata et al., 2006). In order to ensure that the burden-sensing promoter, once activated, confers the cell with a selective growth advantage compared to a non-sensing promoter, it may be necessary to fine-tune the expression level of essential gene. A range of translational strengths can be engineered by varying the translation initiation region introduced with the burden-sensing promoter. Clones with potentially different combinations of candidate burden-sensing promoters are selected and evaluated for maintained protein production over 30-100 generations of cell division. 5.1 Materials and methods Growth medium: YPD medium comprises 1% yeast extract, 2% peptone, 2% glucose. SC medium comprises 6.7 g/L yeast nitrogen base without amino acids and with ammonium sulfate, but lacks uracil. Chromosomal integration and validation of promoter constructs: Constructs for chromosomal integration will contain 300-600 bp upstream of the natively regulated gene. Chromosomal integration of the promoter construct is performed by transformation and homologous recombination using standard electroporation procedures for S. cerevisiae. Correct chromosomal integration of the promoter was validated using colony PCR. Long-term cultivation and production: A single colony from each strain is transferred to 24-deep well plates and cultivated in 1.8 mL YPD medium under conditions inducing recombinant protein production, at 250 rpm and 30 ^C. Following 48 hours of cultivation, cells are passaged to a new deep-well plate under identical conditions by 1000x back- dilution. Samples are analyzed for production using recombinant protein specific assays (e.g. GFP detection) and the cell density is monitored by OD600.   Growth rate measurements of selected strains: Growth rates of individual strains are compared to non-burden addicted production strains. The 96-well plate is sealed with breathe-easy film and growth is measured in a Synergy H1 plate reader for 20 hours at 37°C and 754 rpm linear shaking. OD600 is measured every 10 minutes. 5.2 Results 5.2.1 HAC1-upregulated promoters The HAC1-upregulated promoters comprising an unfolded protein response (UPR) element, e.g. KAR2 (SEQ ID NO.: 91), PDI1 (SEQ ID NO.: 92), SSA1 (SEQ ID NO.: 93) or FPR2 (SEQ ID NO.: 94), are demonstrated to be useful in regulating growth when introduced in front of a native growth regulating gene (e.g. the conditionally essential gene URA3 encoding orotidine 5’-phosphate decarboxylase essential for pyrimidine biosynthesis) in a recombinant protein production Saccharomyces strain producing human insulin precursor or human serum albumin, optionally coupled to GFP. Stability of production is followed under simulated long-term production via serial passages corresponding to 60-80 generations of cell division. In Saccharomyces strains comprising the HAC1-upregulated promoter controlling transcription of the growth- regulating gene (URA3), production is expected to be more stable than the corresponding parent recombinant protein production Saccharomyces strain. 5.3.2 RNA polymerase I upregulated promoters RNA polymerase I transcribe ribosomal RNA genes in yeast. RNA polymerase I upregulated promoters such as promoters of the genes: RPL3 (SEQ ID NO.: 95), RPL6A (SEQ ID NO.: 96) and RPL28 (SEQ ID NO.: 97) are useful in regulating growth of yeast essential genes. Such upregulated promoters are introduced in front of native growth regulating gene (e.g. the conditionally essential gene URA3) in a recombinant protein overproduction strain producing human insulin precursor or human serum albumin, potentially coupled to GFP. Stability of production is followed by experimentally simulated long-term production via serial passages corresponding to 60-80 generations of cell division. In Saccharomyces strains comprising an RNA polymerase I upregulated promoter controlling transcription of the conditionally essential gene, URA3, production will be more stable than the corresponding parent recombinant protein production Saccharomyces strain. 5.3.3 DNA damage responsive promoters DNA damage response in yeast, resulting from heterologous expression broadly induces transcription of the DNA repair system, including OGG1 (SEQ ID NO.: 98), RAD51 (SEQ   ID NO.: 99) and RAD54 (SEQ ID NO.: 100). Promoters of the genes encoding OGG1, RAD51 or RAD54, when operatively linked to an essential gene, are useful for regulating growth of a yeast production cell of the invention. Such promoters are introduced in front of native essential gene (e.g. the growth regulating gene encoding URA3) in cells of a yeast protein production strain producing human insulin precursor or human serum albumin, optionally fused to GFP. Stability of production is followed by under simulated long-term production via serial passages corresponding to 60-80 generations of cell division. In strains with a pRAD51 and/or p_RAD54-upregulated essential gene, production will be more stable. In summary: Burden addiction enhances long-term stability and production in cells of budding yeast engineered to synthesize human serum albumin production or insulin precursor by coupling essential gene transcription to the burden-sensing promoters selected from promoters activated during burden of recombinant protein production or associated with ribosomal RNA promoters (Table 2). Example 6 Examples of burden-addicted yeast strains producing human serum albumin showing enhanced long-term production stability Burden-addiction systems for use in yeast strains include promoters derived from genes encoding: 1) Protein isomerase PDI1 encoding a chaperone belonging to the unfolded protein response of yeasts such as S. cerevisiae and P. pastoris, and whose abundance is frequently upregulated in response to overexpression of a recombinant protein; 2) Ribosomal subunits encoded by the RPL6A and RPL3 genes and 3) FPR2 encoding a peptidyl-prolyl cis-trans isomerase which is known to be activated upon DNA replication stress. Burden-addiction systems based on these promoters were introduced into a strain of Pichia pastoris engineered to express and secrete human serum albumin in order to determine their effect on long term human serum albumin (hSA) production stability. 6.1 Materials and methods: The Pichia pastoris (Komagataella phaffii) strain EGS31 is a derivative of the CBS7435 strain (NRRL-Y11430 or ATCC 76273), engineered to secrete hSA by a genomically integrated cDNA version of the hSA coding ALB1 gene under control of the AOX1 promoter. Strain construction: Burden-addicted versions of the EGS31 strain were generated by genetically integrating a construct comprising a kanMX conditionally selectable G418   resistance gene operably linked to one of the burden-responsive promoters: pPDI1, pFPR2, pRPL3 and pRPL6A. These burden-addiction constructs were integrated into the KU70 genomic locus of the P. pastoris strain by transforming EGS31 cells with linear integration DNA (sequences N1-N3 respectively) flanked by >750 bp homology arms. Transformation was performed using a standard electroporation procedure on exponentially grown cells pretreated with lithium acetate and dithiothreitol (Wu & Letchworth, 2004). Growth Media: BMGY and BMMY liquid media (1L) was prepared as follows: 10 g yeast extract and 20 g peptone was added to 700mL H2O; mixed with a magnetic stirrer and then autoclaved. After cooling to room temperature; the following was added to the solution: 100ml 1M potassium phosphate buffer (pH 6.0); 100 ml 13.4% (w/v) Yeast Nitrogen Base with Ammonium Sulfate without amino acids; 2 ml 0.02% (w/v) biotin, and in the case of BMGY, 100 ml 10% (v/v) glycerol was added; and in the case of BMMY, 100mL 5% (v/v) methanol was added. Cultivation: EGS31 and burden-addicted EGS31 strains were streaked on YPD (1 % yeast extract, 2 % peptone, 2 % D-glucose) agar plates and incubated overnight at 30 deg. C. Single colonies were picked and pre-cultured in 2 mL BMGY where cultures of burden- addicted strains were supplemented 50 ug/mL G418 and cultured at 30 deg. C with 300 rpm horizontal shaking overnight. Expression cultures were seeded using 1 uL pre-   culture into 500 ^L BMMY medium containing different concentrations (0 ^g/mL, 750 ^g/mL) of G418 in a 96-well deep well plate with aerating lid to generate “seed 1”. The cultures were incubated at 30 deg. C for 72 hours (300 rpm horizontal shaking). To generate the next seed, four additional times, grown cultures were serially passaged (500x dilution) to new 500 ^L BMMY medium containing different concentrations (0 ^g/mL, 750 ^g/mL) of G418 in a 96-well deep well plate with aerating lids. At each serial passage, glycerol stocks (20 % glycerol) were stored at -80 deg. C from the grown cultures. To quantify production of secreted hSA, quantification cultures were re-grown from glycerol stocks in 500 ^L BMMY medium containing different concentrations (0 ^g/mL and 750 ^g/mL) of G418 in a 96-well deep well plate with aerating lid for 72 hours. Cultures were centrifuged at 3000 g for 15 minutes and the concentration in 50 ^L supernatants was quantified using a hSA-specific ELISA kit (Abcam catalog no: ab179887: Human Albumin SimpleStep ELISA® Kit) following manufacturer’s instructions. 6.2 Results Strains with a genomically integrated burden addiction promoter, PDI1, operably linked to the selectable kanMX gene displayed higher production of secreted hSA when strain’s burden-addiction system was activated by addition of G418 (figure 13). Further, improved hSA production was seen with higher selection (750 ^g/mL G418), for each of the other tested burden-addiction promoters after approximately 30 cell divisions (Figure 14A – Seed 1). When further cultivated for about an additional 10 cell divisions (Figure 14B –Seed 2) the strains show enhanced levels of hAS production when burden addiction is activated (150-750 ^g/mL G418) compared to the non burden-addicted control and strains without burden addiction activated (0 ^g/mL G418). In conclusion, the cultivation of yeast cells comprising the exemplified burden-addiction systems of the invention, under conditions that activate their respective burden- addiction system, is believed to enrich for high-producing yeast variants within the cultured population. While an increase in hSA production may be detectable after relatively short cultivation (30 cell divisions), enrichment for high-producing yeast variants is both maintained and further enhanced over longer cultivation periods when traditional cultures commonly exhibit a significant decline in productivity.   Example 7 Identification of suitable burden sensing promoter candidates Different engineered production genes and pathways elicit different transcriptional responses indicative of the production process. In order to identify suitable promoter candidates for use as burden sensors, the following experiment was conducted. Methods: Typical genetic escaper cells were isolated from long-term cultivation with the genetically engineered microbial production cells of interest in the intended fermentation medium. Suitable genetic escaper cells are characterized by having at least 5 % higher exponential-phase growth rate and at least 30 % lower production rate or product yield than the original genetically engineered production cell. Production cells and corresponding escaper cells were cultured under intended fermentation conditions, scaled down conditions or shake flask conditions mimicking intended fermentation conditions. At time points corresponding to the highest rate of production in the production cells, samples were taken for RNA sequencing. Total RNA was purified using Purelink RNA Mini kit (Thermo Fischer) and prepared using TruSeq Stranded mRNA kit (Illumina) following the kit manufacturer’s instructions. Reads were mapped and analyzed to the reference genome of the strain and next analyzed for differential expression between the production cells and corresponding escaper cells. Results: Candidate suitable promoters were identified as those driving expression of genes that showed a differential expression of >3 fold higher expression in the production organism relative to at least one isolated genetic escape strain. Example 8: Burden addicted Bacillus subtilis strains producing IgA fragment show enhanced long-term production stability Burden-sensing promoters are shown to be promoters that can sense and be activated by a burden-induced state in a cell resulting from the cell’s synthesis of a recombinant protein, an IgA fragment C-terminally fused to GFP (IgA-GFP). Once activated, burden- sensing promoters are shown to elevate expression of essential gene (operons), iscU or accC, to a level sufficient to confer a selective growth advantage on a cell when compared to a non-productive cell. In order to maximize the dynamic range of essential gene expression in response to its cognate burden sensing promoter, the burden- sensing promoter is randomly combined with variant RBS coding sequences (Table 3.2) conferring different translational strengths. Promoters having burden-sensing properties suitable for use in a burden-addiction genetic circuit are shown to include heat-shock-, DNA damage-, and oxidative stress   response-promoters, as illustrated by the following engineered production strains cultured under simulated large-scale production conditions. 8.1 Materials and Methods Burden-sensing promoters: The promoters, P_hrcA P_perR and their respective integration sequences to target essential genes were generated by PCR and USER cloning resulting in integration vectors (Table 10).   Chromosomal integration of burden-addiction promoters: To construct the candidate burden-addicted strains (Table 11), integration vectors (Table 10) featuring each of the promoters fused to RBS variants (Table 3.2) were integrated by homologous recombination upstream of the operons containing either iscU or accC in the genome of the IgA-GFP producing EGS084 strain (corresponding to B. subtilis KO7 containing the expression cassette of pEG062 inserted in the amyE locus) as follows. Competent cells were prepared according to standard Bacillus transformation methods following a previously described protocol (dx.doi.org/10.17504/protocols.io.bdmti46n) and transformed with pEG151 to pEG166. The transformants were selected on LB agar plates supplemented with spectinomycin (200 µg/mL) and confirmed by colony PCR using the primers E257/E258 (iscU) or E261/E262 (accC). Long term IgA-GFP production assay: A single selected colony from each burden- addicted IgA-GFP producing strain was used to inoculate a 96 deep-well plate containing 500 µL/well Cal18-2 media (dx.doi.org/10.17504/protocols.io.bdmui46w) supplemented with kanamycin (10 µg/mL). The cultures were grown for 24 hours at 30°C/250 RPM horizontally shaking incubator (Innova, 2-inch amplitude). 1 µL of each culture were then transferred (as serial passage) in 499 µL fresh Cal18-2 media (corresponding to   approx. 10 generations per transfer) into a new deep well plate under identical cultivation conditions. These method steps were repeated for a total of up to 13 times. At each serial passage, 100 µL of each strain were banked in equal amount of 50% glycerol for storage and follow-up examinations. To quantify IgA-GFP expression after each passage, cultures were grown for an additional 24 h at the previously described growth conditions. The cultures were spun down at 2000 g for 5 min and washed in 1 volume PBS twice. 20 uL samples of the culture were added to 180 µl PBS (10x dilution) in a 96-well plate and the cell density and IgA-GFP production was quantified by measuring OD600 and GFP fluorescence (λ_ex/ λ_ex = 485nm/528nm) in a Synergy H4 plate reader (Biotek). The specific production level of each culture was quantified as the GFP signal normalized to the OD600 value followed by subtraction of the same value measured in a non GFP-producing Bacillus subtilis KO7 strain. 8.1 Results Following long-term cultivation of five 500-fold dilution serial passages (approx. 65 cell generations), IgA-GFP production was quantified and shown to be improved in the burden addicted strains (Figure 15 and 16). Based on the methodology, similar burden-addicted strains can be constructed using other candidate burden sensors that may match the transcriptional burden response of other production strains. Their suitability can easily be assessed using the devised long- term production assays or for example in continuous small-scale (e.g. 400 mL) production cultures. Example 9: Burden addiction controlling multiple essential genes improves production It is here demonstrated how to introduce two (and by analogy further) burden-sensing promoters controlling different essential gene (operon)s in a single production cell to increase production of desired products and further prolong the period during which the production cells are productive Additionally, by using more than one burden-sensor to control essential genes, the transcriptional space that directs the imposed addiction- based selection regime can be further controlled. The burden-addicted IgA-GFP production strain EGS340 (PhrcA controlling the iscU essential gene operon) was genetically transformed to exchange the native promoter of the accC essential gene operon for the candidate burden sensors PctsR, PdnaK and PhrcA respectively.   9.1 Materials and methods Chromosomal integration of burden-addiction promoters: The spectinomycin resistance marker was exchanged for a chloramphenicol resistance marker in pEG159-pEG166 using the primers E372/E373 (for the vector backbones) and E374/E375 (for the chloramphenicol resistance marker in pDG1662, (Guerout-Fleury et al 1996) to create pEG204 to pEG211 (Table 10). Each of the promoters were integrated upstream of the essential gene operons containing accC in the genome of the EGS340 strain (corresponding to EGS084 containing PperR regulating the iscU essential gene operon) as follows. Competent cells were prepared according to a previously described method (dx.doi.org/10.17504/protocols.io.bdmti46n) and transformed with pEG204 to pEG211. The transformants were selected on LB agar plates supplemented with chloramphenicol (5 µg/mL) and confirmed by colony PCR using the primers E261/E262. Long term IgA-GFP production assay: A single selected colony from each burden- addicted IgA-GFP producing strain was used to inoculate a 96 deep-well plate containing 500 µL/well Cal18-2 media (dx.doi.org/10.17504/protocols.io.bdmui46w) supplemented with kanamycin (10 µg/mL). The cultures were grown for 24 hours at 30°C/250 RPM horizontally shaking incubator (Innova, 2-inch amplitude). 1 µL of each culture were then transferred (as serial passage) in 499 µL fresh Cal18-2 media (corresponding to approx. 10 generations per transfer) into a new deep well plate under identical cultivation conditions. These method steps were repeated for a total of up to 13 times. At each serial passage, 100 µL of each strain were banked in equal amount of 50% glycerol for storage and follow-up examinations. To quantify IgA-GFP expression after each passage, cultures were grown for an additional 24 h at the previously described growth conditions. The cultures were spun down at 2000 g for 5 min and washed in 1 volume PBS twice. 20 uL samples of the culture were added to 180 µl PBS (10x dilution) in a 96-well plate and the cell density and IgA-GFP production was quantified by measuring OD600 and GFP fluorescence (λex/ λex = 485nm/528nm) in a Synergy H4 plate reader (Biotek). The specific production level of each culture was quantified as the   GFP signal normalized to the OD600 value followed by subtraction of the same value measured in a non GFP-producing Bacillus subtilis KO7 strain. Cell disruption and ELISA: Following washing in PBS, the cultures were spun down at 2000g for 5 min. The pellets were resuspended in 1 volume of lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 500 µM EDTA), spun down again at 6000 g for 5 min, and resuspended in 1 volume of lysis buffer supplemented with lysozyme (10 mg/mL). The cells were disrupted by incubating the reactions for 30 min at 37 °C. Afterwards, the reactions were briefly vortexed and spun down at 12000 g for 30 min at 4 °C. The supernatants were subjected to ELISA using the Pig IgA ELISA Kit from Abcam (ab190536) according to manufacturer’s protocol, except that after adding the chromogenic substrate, the absorbance of each well was read at 600 nm every 40 s for 10 min. The relative IgA concentrations were calculated as the slopes of values per OD600 of the original cultures. 9.2 Results The single burden-sensor strain EGS340 carries only the pperR-based burden sensor controlling transcription of the iscU essential gene operon (EGS340). EGS340 is compared to derived strains in which transcription of the accC essential gene operon is also regulated by respectively a pctsR-based burden sensor (EGS460), a pdnaK-based burden sensor (EGS462), and a p_hrcA-based burden sensor (EGS466). Following long- term cultivation for approx. 75 cell generations, IgA-GFP production was quantified and shown to be improved in the burden addicted strains (Figure 17 and 18). Based on the methodology, similar burden-addicted strains can easily be constructed using other candidate burden sensors that may match the transcriptional burden response of other production strains. Their suitability can easily be assessed using the devised long-term production assays or in continuous small-scale (e.g. 400 mL) production cultures. Example 10: Additional method for identification of suitable burden-sensing promoters In this example, suitable candidate burden-sensing promoters are identified for use in burden addiction to sustain long-term heterologous production in particular production strains. The method is divided in an optional track 1 for de novo discovery (RNA-sequencing) and a track 2 for confirmation of putative burden-sensing promoters (q-PCR).   In track 1, using RNA-sequencing the transcriptional activity of coding genes in the production organism during typical production conditions (in terms of e.g. temperature, stirring, product/by-product/substrate concentration/growth phase) is compared to that of a an isolated non/low-producing organism during the same typical production conditions. The culture of the isolated non/low-producing organism may optionally be fed with product to the concentration approximately found in a corresponding culture of the production organism at a given time. The non/low-producing isolate can typically be isolated following culture for >50-100 divisions (preferred), but a suitable isolate strain can also be genetically engineered by genetically inactivating one or several key heterologous production genes. RNA-sequencing is carried out, preferably on at least three replicates, preferably using Illumina short-read sequencing according to standard methods known in the art. Using standard bioinformatical analysis for differential gene expression e.g. incorporating the edgeR work-flow (DOI: 10.18129/B9.bioc.edgeR), suitable candidate burden-sensing promoters are identified from genes up-regulated by at least 5-10 times in the high-producing culture compared to the non/low-producing culture under the same typical production conditions. By identifying candidate burden-sensing promoters using this methodology, promoters activated preferentially by the burden of product formation (compared to merely the stress of production) can be found. Next, in track 2, putative burden-sensing promoters (from track 1 or other lists) are confirmed using q-PCR on samples, similarly comparing one non/low-producer isolates to high-producer isolates during the same typical production conditions (in terms of e.g. temperature, stirring, product/by-product/substrate concentration/growth phase). Suitable candidate burden-sensing promoters are identified from genes up-regulated by at least 5-10 times in the high-producing culture compared to the non/low-producing culture under the same typical production conditions. Example 11: Burden addiction controlling multiple essential genes improves production of recombinant human growth hormone in Escherichia coli The long-term production stability of the burden addicted E. coli strain s7.6#8 was improved by integration of one additional, different burden-sensing promoter controlling transcription of a second essential gene selected e.g. from the list and screened as suggested.   11.1 Materials and methods s7.6#8 single colonies were transformed with a recombineering plasmid such as pKD46 using standard electroporation. Using recombineering, candidate burden-sensing promoters (Table 2) fused to variable RBSs (Table 3.1) were chromosomally integrated directly upstream of the essential gene murI or a similar essential gene, and selected for using spectinomycin resistance gene present in the integration constructs. Following the described steps of recombineering and curing of the recombineering plasmid, five colonies were picked, corresponding to clones with the same promoter integration but having a random variant RBS sequence. Each clone was then transferred to a well of a 96-well plate containing 200 µl 2xYT supplemented with chloramphenicol for maintenance of the plasmid, pMevT, and cultured. 2 µl of each cultured clone was used to validate promotor integration as described in example 1.2.1; prior to freezing the 96-well plate. 11.2 Results The different resulting clones were assessed for long-term production stability by serial passaging of 200x dilutions. Following long-term cultivation for approx. 75 cell generations, IgA-GFP production was quantified and shown to be improved in the new strains compared to the single burden addicted strains only featuring one burden- regulated essential gene. Example 12: Expression-tuning of essential gene CIA1 with TIS sequence variations shows responsive growth in P. pastoris strains This example demonstrates regulation of growth by variations in the TIS (translation initiation site) sequence of the essential gene is demonstrated. The translational strength of the essential gene is modified by such variations in the TIS sequence, which can be used in eukaryotic organisms to titrate the burden-sensing promoter’s burden- response to the expression level of the essential gene similar to the use of ribosomal binding sites in prokaryotic organisms. 12.1 Materials and methods Burden-responsive promoter with TIS (translation initiation site) sequence variations: Variations of promoter, Pgsh2 (promoter of P. pastoris homolog CDS: chr1-4_0496) with different TIS sequences of increasing strength and the cia1 (P. pastoris homolog CDS:   chr1-3_0207) essential gene integration sequence were generated by PCR and USER cloning resulting in integration fragments (Table 14). Chromosomal integration of burden-responsive promoter with TIS sequence variations: To construct the strains with modulated expression of essential gene cia1, integration fragments (Table 14) featuring the promoter, Pgsh2 with TIS sequence variations were integrated by homologous recombination upstream of the cia1 CDS in the IgAL-NanoLuc producing EGS621 strain as follows. Electro-competent cells were prepared according to standard Pichia transformation methods (Wu an Letchworth 2018) and transformed with integration fragments INT1, INT3, INT5, and INT9. Transformants were selected on YPD agar plates supplemented with Zeocin® (50 µg/mL) and confirmed by colony PCR using primers E521 (SEQ ID NO. 237) and E627 (SEQ ID NO. 238).   Pichia TIS sequence growth response assay: Single colonies of the parent strain, EGS621 and the TIS sequence variants EGS1100, EGS1101, EGS1102, and EGS1104 were diluted in 30 µL MilliQ H2O, respectively. 5 µL of diluted colonies were inoculated in 95 µL YPD media to make pre-cultures. EGS621 colonies were supplemented with G418 (50 µg/mL) and the remaining colonies with Zeocin® (50 µg/mL). The pre- cultures were grown overnight at 30 °C and 300 RPM shake. Overnight pre-cultures were then diluted 1000 times in YPD media supplemented with appropriate antibiotic. 1 µL of diluted cultures were inoculated in 99 µL YPD supplemented with appropriate antibiotic in a 96-well culture plate and sealed with a Breathe-Easy® sealing membrane (Sigma-Aldrich, Darmstadt, Germany). The 96-well plate was placed in Synergy 4 plate reader (BioTek, Vermont, USA) and cultures were grown using the following settings: 30 °C, high shaking speed, and OD600 measurement every 10 minutes for 48 hours.12.2 Results A growth difference between all strains is displayed – see figure 19. The parent strain (EGS621) with the native promoter, Pcia1 exhibited the fastest growth. The strains with the TIS sequence variations of the P_gsh2 promoter integrated just upstream of the cia1 CDS (EGS1100, EGS1101, EGS1102, and EGS1104) display growth in accordance with their relative TIS strength. The strain with the strongest TIS sequence, TIS9 (EGS1104) exhibited slightly slower growth than the parent strain (EGS621), and the fastest growth of the TIS strains (EGS1100, EGS1101, EGS1102, and EGS1104). The strain with the weakest TIS sequence, TIS1 (EGS1100) exhibited the slowest growth of all strains. The TIS3 (EGS1101) and TIS5 (EGS1102) strains exhibited growth in accordance with the relative strength between the four TIS sequences. In summary: It is demonstrated that the growth of Pichia strains is responsive to the TIS sequence strength of Pgsh2, which was integrated just upstream of the cia1 CDS, confirming cia1 as being an essential gene for the growth of Pichia strains. Example 13: Implementation of burden addiction in an A. oryzae production strain The burden addiction system of the present invention may also be implemented in other eukaryotes such as fungi. Examples of implementation of burden addiction in an A. oryzae production strain is provided herein. 13.1 Burden sensing promoter element Examples of burden sensing promoters include the promoter sequences (750 bp upstream of start codon) from the gene homologs of bipA (e.g. SEQ ID NO. 189), clxA   (e.g. SEQ ID NO. 190) and agsA (e.g.SEQ ID NO. 239). To construct a burden-addicted A. oryzae production strain, the burden promoters are integrated in front of an essential gene, such as the ERG10 (e.g. SEQ ID NO. 240), PFS2 (e.g. SEQ ID NO. 242) or TUB1 (e.g. SEQ ID NO. 244) homologs. To tune the expression of the essential gene, the burden sensing promoter is integrated with a translation initiation sequence (TIS) library of 4 different variants as presented in table 3.3. These variants replaced the last 6 nucleotides of the promoter sequence, i.e. -6 to –1 relative to the start codon. Examples of burden-addiction integration fragments are presented in table 16. 13.2 Construction of the burden sensing integration construct. To swap the native essential gene promoters for the burden-sensing promoters at three essential gene targeting DNA fragments are prepared by standard molecular cloning. Approximately 1.5 kbp regions up- and down-stream of each essential gene start codons, (respectively ERG10, PFS and TUB1) are amplified from A. oryzae RIB40 genomic DNA. Next, a 750 bp promoter region immediately up-stream of the start codon of respectively bipA, clxA and agsA is likewise amplified from A. oryzae genomic DNA.   Lastly, a synthetic gene fragment containing a selection marker e.g. amdS or pyrG is obtained. All fragments are assembled using e.g. Gibson Assembly technique. From the assembly reaction full length knockout constructs are amplified using PCR. A. oryzae protoplast are prepared according to standard protocols, such as Christensen et al.1988, and transformed with the burden sensing integration constructs and selected on appropriate media. Expression is assayed using shake flasks containing 10 ml YPM medium (2 g/l yeast extract, 2 g/l peptone, and 2% maltose) inoculated with spores from a transformant strain and a reference production strain without the burden sensing promoter element and incubated at 30°C, 200 rpm for 4 days followed by sampling for product. Further assaying in fed batch fermentation is also performed: Tank medium (sucrose 24 g/L, yeast extract 10 g/L, (NH4)2S045 g/L, MgS04-7H202 g/L, K2S02 g/L, citric acid 1 g/L, KH2P0 2 g/L trace metal solution 0.5 ml/L), temperature 34 °C, aeration 1 vvm, and pH is controlled at 6.0 using 10% NH4OH. Medium is inoculated a seed cultivation (spores pre-grown in to shake flask (glycerol 20g/L, yeast extract 18 g/L) for 1 day at 30 °C and 250 rpm) of a transformant strain comprising the burden sensing integration construct and a reference strain. When pH > 6.4, feeding (400 g/L maltose sirup, 1 g/L citric acid) is started at a rate of 3.33 g/L/h. Stirrer speed is controlled to avoid too low (<20%) oxygen tension. References Bonde, M. T., Pedersen, M., Klausen, M. S., Jensen, S. I., Wulff, T., Harrison, S., et al. (2016). Predictable tuning of protein expression in bacteria. Nat. Methods 13. doi:10.1038/nmeth.3727. Christensen, T., Woeldike, H., Boel, E. et al. (1988) High Level Expression of Recombinant Genes in Aspergillus Oryzae. Nat Biotechnol 6, 1419–1422. doi.org/10.1038/nbt1288-1419 Falkenberg et al (2022). Protein expression in Bacillus subtilis. Oct 27, 2022. Website: protocols.io. dx.doi.org/10.17504/protocols.io.bdmti46n Giaever G et al., Nature. 2002 Jul 25;418(6896):387-91. DOI: 10.1038/nature00935 Jain, R., Kumar, P. & Varshney, U. A distinct role of formamidopyrimidine DNA glycosylase (MutM) in down-regulation of accumulation of G, C mutations and protection against oxidative stress in mycobacteria. DNA Repair (Amst). 6, 1774–1785 (2007).   Guerout-Fleury AM, Frandson N, Stragier P. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57–61 Koskiniemi, S., Pränting, M., Gullberg, E., Näsvall, J., & Andersson, D. I. (2011). Activation of cryptic aminoglycoside resistance in Salmonella enterica. Molecular Microbiology, Vol. 80, pp. 1464–1478. https://doi.org/10.1111/j.1365- 2958.2011.07657.x Kimata, Y., Ishiwata-Kimata, Y., Yamada, S., and Kohno, K. (2006). Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins. Genes to Cells 11, 59–69. doi:10.1111/j.1365- 2443.2005.00921.x. Laferté, A., Favry, E., Sentenac, A., Riva, M., Carles, C., and Chédin, S. (2006). The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components. Genes Dev. 20, 2030–2040. doi:10.1101/gad.386106. Maeda, M., Shimada, T., and Ishihama, A. (2015). Strength and Regulation of Seven rRNA Promoters in Escherichia coli. PLoS One 10, 1–19. doi:10.1371/journal.pone.0144697. Nonaka, G., Blankschien, M., Herman, C., Gross, C. a, and Rhodius, V. a (2006). Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress. Genes Dev. 20, 1776–89. doi:10.1101/gad.1428206. Pitera, D. J., Paddon, C. J., Newman, J. D., and Keasling, J. D. (2007). Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab. Eng. 9, 193-207. doi:10.1016/j.ymben.2006.11.002. Rugbjerg, P., Sarup-Lytzen, K., Nagy, M., and Sommer, M. O. A. (2018). Synthetic addiction extends the productive life time of engineered Escherichia coli populations. Proc. Natl. Acad. Sci. 115, 2347–2352. doi:10.1073/pnas.1718622115. Wu, S. and Letchworth, G.J. (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36, 152– 154 Wu an Letchworth (2018) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. BIOTECHNIQUESVOL.36, NO. 1DRUG DISCOVERY AND GENOMIC TECHNOLOGIES. doi.org/10.2144/04361DD02. Yoon, S. H., Han, M. J., Lee, S. Y., Jeong, K. J., and Yoo, J. S. (2003). Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture. Biotechnol. Bioeng. 81, 753–767. doi:10.1002/bit.10626. Uppal, S., Shetty, D. M., & Jawali, N. (2014). Cyclic AMP receptor protein regulates cspd, a bacterial toxin gene, in Escherichia coli. Journal of Bacteriology, 196(8), 1569–1577. https://doi.org/10.1128/JB.01476-13   Yamanaka, K., & Inouye, M. (1997). Growth-phase-dependent expression of cspD, encoding a member of the CspA family in Escherichia coli. Journal of Bacteriology, 179(16), 5126–5130. https://doi.org/10.1128/jb.179.16.5126-5130.1997

SEQUENCE LISTING

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<400> 1 atg aaa ctc ttt gcc cag ggt act tca ctg gac ctt agc cat cct cac 48 Met Lys Leu Phe Ala Gin Gly Thr Ser Leu Asp Leu Ser His Pro His 1 5 10 15 gta atg ggg ate ctc aac gtc acg cct gat tec ttt tcg gat ggt ggc 96 Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser Asp Gly Gly 20 25 30 acg cat aac tcg ctg ata gat gcg gtg aaa cat gcg aat ctg atg atc 144 Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn Leu Met Ile 35 40 45 aat gct ggc gcg acg atc att gac gtt ggt ggc gag tec acg cgc cca 192 Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser Thr Arg Pro 50 55 60 ggg gcg gcg gaa gtt agc gtt gaa gaa gag ttg caa cgt gtt att cct 240 Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gin Arg Val Ile Pro 65 70 75 80 gtg gtt gag gca att gct caa cgc ttc gaa gtc tgg atc tcg gtc gat 288 Val Val Glu Ala Ile Ala Gin Arg Phe Glu Val Trp Ile Ser Val Asp 85 90 95 aca tec aaa cca gaa gtc atc cgt gag tca gcg aaa gtt ggc gct cac 336 Thr Ser Lys Pro Glu Val Ile Arg Glu Ser Ala Lys Val Gly Ala His 100 105 110 att att aat gat atc cgc tec ctt tec gaa cct ggc gct ctg gag gcg 384 Ile Ile Asn Asp Ile Arg Ser Leu Ser Glu Pro Gly Ala Leu Glu Ala 115 120 125 gct gca gaa acc ggt tta ccg gtt tgt ctg atg cat atg cag gga aat 432 Ala Ala Glu Thr Gly Leu Pro Val Cys Leu Met His Met Gin Gly Asn 130 135 140 cca aaa acc atg cag gaa gct ccg aag tat gac gat gtc ttt gca gaa 480

Pro Lys Thr Met Gin Glu Ala Pro Lys Tyr Asp Asp Val Phe Ala Glu

145 150 155 160 gtg aat cgc tac ttt att gag caa ata gca cgt tgc gag cag gcg ggt 528 Val Asn Arg Tyr Phe Ile Glu Gin Ile Ala Arg Cys Glu Gin Ala Gly

165 170 175 atc gca aaa gag aaa ttg ttg ctc gac ccc gga ttc ggt ttc ggt aaa 576 Ile Ala Lys Glu Lys Leu Leu Leu Asp Pro Gly Phe Gly Phe Gly Lys

180 185 190 aat ctc tec cat aac tat tca tta ctg gcg cgc ctg gct gaa ttt cac 624

Asn Leu Ser His Asn Tyr Ser Leu Leu Ala Arg Leu Ala Glu Phe His

195 200 205 cat ttc aac ctg ccg ctg ttg gtg ggt atg tca cga aaa tcg atg att 672

His Phe Asn Leu Pro Leu Leu Val Gly Met Ser Arg Lys Ser Met Ile

210 215 220 ggg cag ctg ctg aac gtg ggg ccg tcc gag cgc ctg agc ggt agt ctg 720

Gly Gin Leu Leu Asn Val Gly Pro Ser Glu Arg Leu Ser Gly Ser Leu

225 230 235 240 gcc tgt gcg gtc att gcc gca atg caa ggc gcg cac atc att cgt gtt 768

Ala Cys Ala Val Ile Ala Ala Met Gin Gly Ala His Ile Ile Arg Val

245 250 255 cat gac gtc aaa gaa acc gta gaa gcg atg egg gtg gtg gaa gcc act 816

His Asp Val Lys Glu Thr Val Glu Ala Met Arg Val Val Glu Ala Thr

260 265 270 ctg tct gca aag gaa aac aaa cgc tat gag taa tcgtaaatat ttcggtaeeg 869

Leu Ser Ala Lys Glu Asn Lys Arg Tyr Glu

275 280 atgggattcg tggtcgtgta ggggatgcgc cgatcacacc tgattttgtg cttaagctgg 929 gttgggccgc gggtaaagtg ctggcgcgcc acggctcccg taagatcatt attggtaaag 989 acacgcgtat ttctggctat atgctggagt cagcactgga agcgggtctg gcggcagcgg 1049 gcctttccgc actcttcact ggcccgatgc caacaccggc cgtggcttat etgaegcgta 1109 ccttccgcgc agaggccgga attgtgatat ctgcatcgca taacccgttc tacgataatg 1169 gcattaaatt cttctctatc gacggcacca aaetgccgga tgcggtagaa gaggccatcg 1229 aagcggaaat ggaaaaggag atcagctgcg ttgattcgge agaactgggt aaagccagcc 1289 gtatcgttga tgccgcgggt egctatatcg agttttgcaa agccacgttc ccgaacgaac 1349 ttagcctcag tgaaetgaag attgtggtgg attgtgcaaa cggtgcgact tatcacatcg 1409 cgccgaacgt gctgcgcgaa ctgggggcga aegttatcge tatcggttgt gagccaaacg 1469 gtgtaaacat caatgccgaa gtgggggcta ccgacgttcg cgcgctccag gctcgtgtgc 1529 tggctgaaaa agcggatctc ggtattgcct tcgacggcga tggcgatcgc gtgattatgg 1589 ttgaccatga aggcaataaa gtcgatggcg atcagatcat gtatatcatc gcgcgtgaag 1649 gtcttcgtca gggccagctg cgtggtggcg ctgtgggtac attgatgagc aacatggggc 1709 ttgaactggc gctgaaacag ttaggaattc catttgcgcg cgcgaaagtg ggtgaccgct 1769 acgtactgga aaaaatgcag gagaaaggct ggcgtatcgg tgcagagaat tccggtcatg 1829 tgatcctgct ggataaaact actaccggtg acggcatcgt tgctggcttg caggtgctgg 1889 cggctatggc acgtaaccat atgagcctgc acgacctttg cagcggcatg aaaatgttcc 1949 cgcagattct ggttaacgta cgttacaccg caggtagcgg cgatccactt gagcatgagt 2009 cagttaaagc cgtgaccgca gaggttgaag ctgcgctggg caaccgtgga cgcgtgttgc 2069 tgcgtaaatc cggcaccgaa ccgttaattc gcgtgatggt ggaaggcgaa gacgaagcgc 2129 aggtgactga atttgcacac cgcatcgccg atgcagtaaa agccgtttaa 2179

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Met Lys Leu Phe Ala Gin Gly Thr Ser Leu Asp Leu Ser His Pro His 1 5 10 15 Val Met Gly Ile Leu Asn Val Thr Pro Asp Ser Phe Ser Asp Gly Gly 20 25 30

Thr His Asn Ser Leu Ile Asp Ala Val Lys His Ala Asn Leu Met Ile 35 40 45

Asn Ala Gly Ala Thr Ile Ile Asp Val Gly Gly Glu Ser Thr Arg Pro 50 55 60

Gly Ala Ala Glu Val Ser Val Glu Glu Glu Leu Gin Arg Val Ile Pro 65 70 75 80 Val Val Glu Ala Ile Ala Gin Arg Phe Glu Val Trp Ile Ser Val Asp 85 90 95

Thr Ser Lys Pro Glu Val Ile Arg Glu Ser Ala Lys Val Gly Ala His 100 105 110 Ile Ile Asn Asp Ile Arg Ser Leu Ser Glu Pro Gly Ala Leu Glu Ala

115 120 125

Ala Ala Glu Thr Gly Leu Pro Val Cys Leu Met His Met Gin Gly Asn 130 135 140

Pro Lys Thr Met Gin Glu Ala Pro Lys Tyr Asp Asp Val Phe Ala Glu 145 150 155 160 Val Asn Arg Tyr Phe Ile Glu Gin Ile Ala Arg Cys Glu Gin Ala Gly

165 170 175 Ile Ala Lys Glu Lys Leu Leu Leu Asp Pro Gly Phe Gly Phe Gly Lys

180 185 190

Asn Leu Ser His Asn Tyr Ser Leu Leu Ala Arg Leu Ala Glu Phe His 195 200 205

His Phe Asn Leu Pro Leu Leu Val Gly Met Ser Arg Lys Ser Met Ile

210 215 220

Gly Gin Leu Leu Asn Val Gly Pro Ser Glu Arg Leu Ser Gly Ser Leu 225 230 235 240

Ala Cys Ala Val Ile Ala Ala Met Gin Gly Ala His Ile Ile Arg Val

245 250 255

His Asp Val Lys Glu Thr Val Glu Ala Met Arg Val Val Glu Ala Thr 260 265 270

Leu Ser Ala Lys Glu Asn Lys Arg Tyr Glu 275 280

<210> 3

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<220>

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<223> E . coli essential gene glmM

<400> 3 atg agt aat cgt aaa tat ttc ggt acc gat ggg att cgt ggt cgt gta 48 Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 ggg gat gcg ccg atc aca cct gat ttt gtg ctt aag ctg ggt tgg gcc 96

Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala

20 25 30 gcg ggt aaa gtg ctg gcg cgc cac ggc tcc cgt aag att att att ggt 144

Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly

35 40 45 aaa gac acg cgt att tct ggc tat atg ctg gag tca gca ctg gaa gcg 192

Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala

50 55 60 ggt ctg gcg gca gcg ggc ctt tcc gca ctc ttc act ggc ccg atg cca 240

Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro

65 70 75 80 aca ccg gcc gtg gct tat ctg acg cgt acc ttc cgc gca gag gcc gga 288

Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly

85 90 95 att gtg ata tct gca tcg cat aac ccg ttc tac gat aat ggc att aaa 336 Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys

100 105 110 ttc ttc tct atc gac ggc acc aaa ctg ccg gat gcg gta gaa gag gcc 384

Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala

115 120 125 atc gaa gcg gaa atg gaa aag gag atc agc tgc gtt gat tcg gca gaa 432 Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu

130 135 140 ctg ggt aaa gcc agc cgt atc gtt gat gcc gcg ggt cgc tat atc gag 480

Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu

145 150 155 160 ttt tgc aaa gcc acg ttc ccg aac gaa ctt agc ctc agt gaa ctg aag 528

Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys

165 170 175 att gtg gtg gat tgt gca aac ggt gcg act tat cac atc gcg ccg aac 576 Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn

180 185 190 gtg ctg cgc gaa ctg ggg gcg aac gtt atc gct atc ggt tgt gag cca 624 Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro

195 200 205 aac ggt gta aac atc aat gcc gaa gtg ggg gct acc gac gtt cgc gcg 672

Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala

210 215 220 ctc cag gct cgt gtg ctg gct gaa aaa gcg gat ctc ggt att gcc ttc 720

Leu Gin Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe

225 230 235 240 gac ggc gat ggc gat cgc gtg att atg gtt gac cat gaa ggc aat aaa 768

Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys

245 250 255 gtc gat ggc gat cag atc atg tat atc atc gcg cgt gaa ggt ctt cgt 816 Val Asp Gly Asp Gin Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg

260 265 270 cag ggc cag ctg cgt ggt ggc gct gtg ggt aca ttg atg agc aac atg 864

Gin Gly Gin Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met

275 280 285 ggg ctt gaa ctg gcg ctg aaa cag tta gga att cca ttt gcg cgc gcg 912

Gly Leu Glu Leu Ala Leu Lys Gin Leu Gly Ile Pro Phe Ala Arg Ala

290 295 300 aaa gtg ggt gac cgc tac gta ctg gaa aaa atg cag gag aaa ggc tgg 960

Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gin Glu Lys Gly Trp

305 310 315 320 cgt atc ggt gca gag aat tcc ggt cat gtg atc ctg ctg gat aaa act 1008

Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr

325 330 335 act acc ggt gac ggc atc gtt gct ggc ttg cag gtg ctg gcg gcg atg 1056

Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gin Val Leu Ala Ala Met

340 345 350 gca cgt aac cat atg agc ctg cac gac ctt tgc agc ggc atg aaa atg 1104

Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met

355 360 365 ttc ccg cag att ctg gtt aac gta cgt tac acc gca ggt agc ggc gat 1152

Phe Pro Gin Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp

370 375 380 cca ctt gag cat gag tca gtt aaa gcc gtg acc gca gag gtt gaa gct 1200

Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala

385 390 395 400 gcg ctg ggc aac cgt gga cgc gtg ttg ctg cgt aaa tcc ggc acc gaa 1248

Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu

405 410 415 ccg tta att cgc gtg atg gtg gaa ggc gaa gac gaa gcg cag gtg act 1296

Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gin Val Thr

420 425 430 gaa ttt gca cac cgc atc gcc gat gca gta aaa gcc gtt taa 1338

Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val

435 440 445

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Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val

1 5 10 15

Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20 25 30

Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45

Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60

Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys

100 105 110

Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu

130 135 140

Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160

Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys

165 170 175 Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn

180 185 190 Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205

Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala

210 215 220

Leu Gin Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240

Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys

245 250 255 Val Asp Gly Asp Gin Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270

Gin Gly Gin Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285

Gly Leu Glu Leu Ala Leu Lys Gin Leu Gly Ile Pro Phe Ala Arg Ala

290 295 300

Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gin Glu Lys Gly Trp 305 310 315 320

Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335

Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gin Val Leu Ala Ala Met 340 345 350

Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365

Phe Pro Gin Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp

370 375 380

Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400

Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu

405 410 415

Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gin Val Thr 420 425 430

Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val

435 440 445

<210> 5

<211> 858

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<222> (1) . . (858)

<223> E . coli essential gene murl

<400> 5 atg gct acc aaa ctg cag gac ggg aat aca cct tgt ctg gca gct aca 48

Met Ala Thr Lys Leu Gin Asp Gly Asn Thr Pro Cys Leu Ala Ala Thr

1 5 10 15 cct tct gaa cca cgt ccc acc gtg ctg gtg ttt gac tec ggc gtc ggt 96

Pro Ser Glu Pro Arg Pro Thr Val Leu Val Phe Asp Ser Gly Val Gly

20 25 30 ggg ttg tcg gtc tat gac gag atc egg cat ctc tta ccg gat ctc cat 144

Gly Leu Ser Val Tyr Asp Glu Ile Arg His Leu Leu Pro Asp Leu His

35 40 45 tac att tat gct ttc gat aac gtc gct ttc ccg tat ggc gaa aaa agc 192

Tyr Ile Tyr Ala Phe Asp Asn Val Ala Phe Pro Tyr Gly Glu Lys Ser

50 55 60 gaa gcg ttt att gtt gag cga gtg gtg gca att gtc acc gcg gtg caa 240

Glu Ala Phe Ile Val Glu Arg Val Val Ala Ile Val Thr Ala Val Gin

65 70 75 80 gaa cgt tat ccc ctt gcg ctg gct gtg gtc gct tgc aac act gcc agt 288 Glu Arg Tyr Pro Leu Ala Leu Ala Val Val Ala Cys Asn Thr Ala Ser 85 90 95 acc gtt tca ctt cct gca tta cgc gaa aag ttc gac ttc ccg gtt gtt 336

Thr Val Ser Leu Pro Ala Leu Arg Glu Lys Phe Asp Phe Pro Val Val

100 105 110 ggt gtc gtg ccg gcg att aaa cct gct gca cgt ctg acg gca aat ggc 384

Gly Val Val Pro Ala Ile Lys Pro Ala Ala Arg Leu Thr Ala Asn Gly

115 120 125 att gtc gga tta ctg gca acc cgc gga aca gtt aaa cgt tct tat act 432 Ile Val Gly Leu Leu Ala Thr Arg Gly Thr Val Lys Arg Ser Tyr Thr

130 135 140 cat gag ctg atc gcg cgt ttc gct aat gaa tgc cag ata gaa atg ctg 480

His Glu Leu Ile Ala Arg Phe Ala Asn Glu Cys Gin Ile Glu Met Leu

145 150 155 160 ggc tcg gca gag atg gtt gag ttg gct gaa gcg aag cta cat ggc gaa 528

Gly Ser Ala Glu Met Val Glu Leu Ala Glu Ala Lys Leu His Gly Glu

165 170 175 gat gtt tct ctg gat gca cta aaa cgt atc cta cgc ccg tgg tta aga 576

Asp Val Ser Leu Asp Ala Leu Lys Arg Ile Leu Arg Pro Trp Leu Arg

180 185 190 atg aaa gag ccg cca gat acc gtt gta ttg ggt tgc acc cat ttc cct 624

Met Lys Glu Pro Pro Asp Thr Val Val Leu Gly Cys Thr His Phe Pro

195 200 205 cta cta caa gaa gaa ctg tta caa gtg ctg cca gag gga acc egg ctg 672

Leu Leu Gin Glu Glu Leu Leu Gin Val Leu Pro Glu Gly Thr Arg Leu

210 215 220 gtg gat tct ggc gca gcg att gct cgc cga acg gcc tgg ttg tta gaa 720 Val Asp Ser Gly Ala Ala Ile Ala Arg Arg Thr Ala Trp Leu Leu Glu

225 230 235 240 cat gaa gcc ccg gat gca aaa tct gcc gat gcg aat att gcc ttt tgt 768

His Glu Ala Pro Asp Ala Lys Ser Ala Asp Ala Asn Ile Ala Phe Cys

245 250 255 atg gca atg acg cca gga gct gaa caa tta ttg ccc gtt tta cag cgt 816

Met Ala Met Thr Pro Gly Ala Glu Gin Leu Leu Pro Val Leu Gin Arg

260 265 270 tac ggc ttc gaa acg ctc gaa aaa ctg gca gtt tta ggc tga 858

Tyr Gly Phe Glu Thr Leu Glu Lys Leu Ala Val Leu Gly

275 280 285

<210> 6

<211> 285

<212> PRT

<213> E . coli

<400> 6

Met Ala Thr Lys Leu Gin Asp Gly Asn Thr Pro Cys Leu Ala Ala Thr 1 5 10 15 Pro Ser Glu Pro Arg Pro Thr Val Leu Val Phe Asp Ser Gly Val Gly 20 25 30

Gly Leu Ser Val Tyr Asp Glu Ile Arg His Leu Leu Pro Asp Leu His 35 40 45

Tyr Ile Tyr Ala Phe Asp Asn Val Ala Phe Pro Tyr Gly Glu Lys Ser 50 55 60

Ala Phe Ile Val Glu Arg Val Val Ala Ile Val Thr Ala Val Gin 70 75 80

Glu Arg Tyr Pro Leu Ala Leu Ala Val Val Ala Cys Asn Thr Ala Ser 85 90 95

Thr Val Ser Leu Pro Ala Leu Arg Glu Lys Phe Asp Phe Pro Val Val 100 105 110

Gly Val Val Pro Ala Ile Lys Pro Ala Ala Arg Leu Thr Ala Asn Gly 115 120 125 Ile Val Gly Leu Leu Ala Thr Arg Gly Thr Val Lys Arg Ser Tyr Thr 130 135 140

His Glu Leu Ile Ala Arg Phe Ala Asn Glu Cys Gin Ile Glu Met Leu 145 150 155 160

Gly Ser Ala Glu Met Val Glu Leu Ala Glu Ala Lys Leu His Gly Glu 165 170 175

Asp Val Ser Leu Asp Ala Leu Lys Arg Ile Leu Arg Pro Trp Leu Arg 180 185 190

Met Lys Glu Pro Pro Asp Thr Val Val Leu Gly Cys Thr His Phe Pro 195 200 205

Leu Leu Gin Glu Glu Leu Leu Gin Val Leu Pro Glu Gly Thr Arg Leu 210 215 220

Val Asp Ser Gly Ala Ala Ile Ala Arg Arg Thr Ala Trp Leu Leu Glu 225 230 235 240

His Glu Ala Pro Asp Ala Lys Ser Ala Asp Ala Asn Ile Ala Phe Cys 245 250 255

Met Ala Met Thr Pro Gly Ala Glu Gin Leu Leu Pro Val Leu Gin Arg 260 265 270 Tyr Gly Phe Glu Thr Leu Glu Lys Leu Ala Val Leu Gly 275 280 285

<210> 7

<211> 1104

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1104 )

<223> E . coli essential gene asd

<400> 7 atg aaa aat gtt ggt ttt atc ggc tgg cgc ggt atg gtc ggc tcc gtt 48 Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val 1 5 10 15 ctc atg caa cgc atg gtt gaa gag cgc gac ttc gac gcc att cgc cct 96

Leu Met Gin Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro

20 25 30 gtc tac ttt tct act tct cag ctt ggt cag gct gcg ccg tct ttt ggc 144 Val Tyr Phe Ser Thr Ser Gin Leu Gly Gin Ala Ala Pro Ser Phe Gly

35 40 45 gga acc act ggc aca ctt cag gat gcc ttt gat ctg gag gcg cta aag 192

Gly Thr Thr Gly Thr Leu Gin Asp Ala Phe Asp Leu Glu Ala Leu Lys

50 55 60 gcc ctc gat atc att gtg acc tgt cag ggc ggc gat tat acc aac gaa 240

Ala Leu Asp Ile Ile Val Thr Cys Gin Gly Gly Asp Tyr Thr Asn Glu

65 70 75 80 atc tat cca aag ctt cgt gaa agc gga tgg cag ggt tac tgg att gac 288 Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gin Gly Tyr Trp Ile Asp

85 90 95 gca gca tcg tct ctg cgc atg aaa gat gac gcc atc atc att ctt gac 336

Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp

100 105 110 ccc gtc aat cag gac gtc att acc gac gga tta aat aat ggc atc agg 384

Pro Val Asn Gin Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg

115 120 125 act ttt gtt ggc ggt aac tgt acc gta agc ctg atg ttg atg tcg ttg 432

Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu

130 135 140 ggt ggt tta ttc gcc aat gat ctt gtt gat tgg gtg tcc gtt gca acc 480

Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr

145 150 155 160 tac cag gcc gct tcc ggc ggt ggt gcg cga cat atg cgt gag tta tta 528

Tyr Gin Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu

165 170 175 acc cag atg ggc cat ctg tat ggc cat gta gca gat gaa ctc gcg acc 576 Thr Gin Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr

180 185 190 ccg tcc tct gct att ctc gat atc gaa cgc aaa gtc aca acc tta acc 624

Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr

195 200 205 cgt agc ggt gag ctg ccg gtg gat aac ttt ggc gtg ccg ctg gcg ggt 672

Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly

210 215 220 agc ctg att ccg tgg atc gac aaa cag ctc gat aac ggt cag agc cgc 720

Ser Leu Ile Pro Trp Ile Asp Lys Gin Leu Asp Asn Gly Gin Ser Arg

225 230 235 240 gaa gag tgg aaa ggg cag gcg gaa acc aac aag atc ctc aac aca tct 768

Glu Glu Trp Lys Gly Gin Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser

245 250 255 tec gta att ccg gta gat ggt tta tgt gtg cgt gtc ggg gca ttg cgc 816

Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg

260 265 270 tgc cac agc cag gca ttc act att aaa ttg aaa aaa gat gtg tcg att 864

Cys His Ser Gin Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile

275 280 285 ccg acc gtg gaa gaa ctg ctg gct gcg cac aat ccg tgg gcg aaa gtc 912

Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val

290 295 300 gtt ccg aac gat egg gaa atc act atg cgt gag cta acc cca gct gcc 960 Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala

305 310 315 320 gtt acc ggc acg ctg acc acg ccg gta ggc cgc ctg cgt aag ctg aat 1008 Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn

325 330 335 atg gga cca gag ttc ctg tca gcc ttt acc gtg ggc gac cag ctg ctg 1056

Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gin Leu Leu

340 345 350 tgg ggg gcc gcg gag ccg ctg cgt egg atg ctt cgt caa ctg gcg taa 1104

Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gin Leu Ala

355 360 365

<210> 8

<211> 367

<212> PRT

<213> E . coli

<400> 8

Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val

1 5 10 15

Leu Met Gin Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro 20 25 30 Val Tyr Phe Ser Thr Ser Gin Leu Gly Gin Ala Ala Pro Ser Phe Gly 35 40 45

Gly Thr Thr Gly Thr Leu Gin Asp Ala Phe Asp Leu Glu Ala Leu Lys 50 55 60

Ala Leu Asp Ile Ile Val Thr Cys Gin Gly Gly Asp Tyr Thr Asn Glu 65 70 75 80 Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gin Gly Tyr Trp Ile Asp

85 90 95

Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp 100 105 110

Pro Val Asn Gin Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg 115 120 125

Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu

130 135 140

Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr 145 150 155 160

Tyr Gin Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu

165 170 175

Thr Gin Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr 180 185 190

Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr 195 200 205

Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly

210 215 220

Ser Leu Ile Pro Trp Ile Asp Lys Gin Leu Asp Asn Gly Gin Ser Arg 225 230 235 240

Glu Glu Trp Lys Gly Gin Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser

245 250 255

Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg 260 265 270

Cys His Ser Gin Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile 275 280 285 Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val

290 295 300 Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala 305 310 315 320 Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn

325 330 335

Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gin Leu Leu 340 345 350

Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gin Leu Ala 355 360 365

<210> 9

<211> 1104

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1104 )

<223> E . coll essential gene thyA

<400> 9 atg aaa aat gtt ggt ttt atc ggc tgg cgc ggt atg gtc ggc tec gtt 48 Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val 1 5 10 15 ctc atg caa cgc atg gtt gaa gag cgc gac ttc gac gcc att cgc cct 96 Leu Met Gin Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro 20 25 30 gtc tac ttt tct act tct cag ctt ggt cag gct gcg ccg tct ttt ggc 144 Val Tyr Phe Ser Thr Ser Gin Leu Gly Gin Ala Ala Pro Ser Phe Gly 35 40 45 gga acc act ggc aca ctt cag gat gcc ttt gat ctg gag gcg cta aag 192 Gly Thr Thr Gly Thr Leu Gin Asp Ala Phe Asp Leu Glu Ala Leu Lys 50 55 60 gcc ctc gat atc att gtg acc tgt cag ggc ggc gat tat acc aac gaa 240 Ala Leu Asp Ile Ile Val Thr Cys Gin Gly Gly Asp Tyr Thr Asn Glu 65 70 75 80 atc tat cca aag ctt cgt gaa agc gga tgg cag ggt tac tgg att gac 288 Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gin Gly Tyr Trp Ile Asp 85 90 95 gca gca tcg tct ctg cgc atg aaa gat gac gcc atc atc att ctt gac 336 Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp 100 105 110 ccc gtc aat cag gac gtc att acc gac gga tta aat aat ggc atc agg 384 Pro Val Asn Gin Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg 115 120 125 act ttt gtt ggc ggt aac tgt acc gta agc ctg atg ttg atg tcg ttg 432 Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu 130 135 140 ggt ggt tta ttc gcc aat gat ctt gtt gat tgg gtg tcc gtt gca acc 480

Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr

145 150 155 160 tac cag gcc gct tcc ggc ggt ggt gcg cga cat atg cgt gag tta tta 528

Tyr Gin Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu

165 170 175 acc cag atg ggc cat ctg tat ggc cat gta gca gat gaa ctc gcg acc 576

Thr Gin Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr

180 185 190 ccg tcc tct gct att ctc gat atc gaa cgc aaa gtc aca acc tta acc 624

Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr

195 200 205 cgt agc ggt gag ctg ccg gtg gat aac ttt ggc gtg ccg ctg gcg ggt 672

Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly

210 215 220 agc ctg att ccg tgg atc gac aaa cag ctc gat aac ggt cag agc cgc 720

Ser Leu Ile Pro Trp Ile Asp Lys Gin Leu Asp Asn Gly Gin Ser Arg

225 230 235 240 gaa gag tgg aaa ggg cag gcg gaa acc aac aag atc ctc aac aca tct 768

Glu Glu Trp Lys Gly Gin Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser

245 250 255 tcc gta att ccg gta gat ggt tta tgt gtg cgt gtc ggg gca ttg cgc 816

Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg

260 265 270 tgc cac agc cag gca ttc act att aaa ttg aaa aaa gat gtg tcg att 864

Cys His Ser Gin Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile

275 280 285 ccg acc gtg gaa gaa ctg ctg gct gcg cac aat ccg tgg gcg aaa gtc 912

Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val

290 295 300 gtt ccg aac gat egg gaa atc act atg cgt gag cta acc cca gct gcc 960 Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala

305 310 315 320 gtt acc ggc acg ctg acc acg ccg gta ggc cgc ctg cgt aag ctg aat 1008 Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn

325 330 335 atg gga cca gag ttc ctg tca gcc ttt acc gtg ggc gac cag ctg ctg 1056

Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gin Leu Leu

340 345 350 tgg ggg gcc gcg gag ccg ctg cgt egg atg ctt cgt caa ctg gcg taa 1104

Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gin Leu Ala

355 360 365 <210> 10

<211> 367

<212> PRT

<213> E . coli

<400> 10

Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val

1 5 10 15

Leu Met Gin Arg Met Val Glu Glu Arg Asp Phe Asp Ala Ile Arg Pro 20 25 30 Val Tyr Phe Ser Thr Ser Gin Leu Gly Gin Ala Ala Pro Ser Phe Gly 35 40 45

Gly Thr Thr Gly Thr Leu Gin Asp Ala Phe Asp Leu Glu Ala Leu Lys 50 55 60

Ala Leu Asp Ile Ile Val Thr Cys Gin Gly Gly Asp Tyr Thr Asn Glu 65 70 75 80 Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gin Gly Tyr Trp Ile Asp 85 90 95

Ala Ala Ser Ser Leu Arg Met Lys Asp Asp Ala Ile Ile Ile Leu Asp 100 105 110

Pro Val Asn Gin Asp Val Ile Thr Asp Gly Leu Asn Asn Gly Ile Arg 115 120 125

Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu 130 135 140

Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr

145 150 155 160

Tyr Gin Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu 165 170 175

Thr Gin Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr 180 185 190

Pro Ser Ser Ala Ile Leu Asp Ile Glu Arg Lys Val Thr Thr Leu Thr 195 200 205

Arg Ser Gly Glu Leu Pro Val Asp Asn Phe Gly Val Pro Leu Ala Gly 210 215 220 Ser Leu Ile Pro Trp Ile Asp Lys Gin Leu Asp Asn Gly Gin Ser Arg 225 230 235 240

Glu Glu Trp Lys Gly Gin Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser

245 250 255

Ser Val Ile Pro Val Asp Gly Leu Cys Val Arg Val Gly Ala Leu Arg 260 265 270

Cys His Ser Gin Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile 275 280 285

Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val

290 295 300 Val Pro Asn Asp Arg Glu Ile Thr Met Arg Glu Leu Thr Pro Ala Ala 305 310 315 320 Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn

325 330 335

Met Gly Pro Glu Phe Leu Ser Ala Phe Thr Val Gly Asp Gin Leu Leu 340 345 350

Trp Gly Ala Ala Glu Pro Leu Arg Arg Met Leu Arg Gin Leu Ala 355 360 365

<210> 11

<211> 1842

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1842 )

<223> E . coli essential gene rpoD

<400> 11 atg gag caa aac ccg cag tca cag ctg aaa ctt ctt gtc acc cgt ggt 48 Met Glu Gin Asn Pro Gin Ser Gin Leu Lys Leu Leu Val Thr Arg Gly 1 5 10 15 aag gag caa ggc tat ctg acc tat gcc gag gtc aat gac cat ctg ccg 96

Lys Glu Gin Gly Tyr Leu Thr Tyr Ala Glu Val Asn Asp His Leu Pro

20 25 30 gaa gat atc gtc gat tca gat cag atc gaa gac atc atc caa atg atc 144

Glu Asp Ile Val Asp Ser Asp Gin Ile Glu Asp Ile Ile Gin Met Ile

35 40 45 aac gac atg ggc att cag gtg atg gaa gaa gca ccg gat gcc gat gat 192

Asn Asp Met Gly Ile Gin Val Met Glu Glu Ala Pro Asp Ala Asp Asp

50 55 60 ctg atg ctg gct gaa aac acc gcg gac gaa gat gct gcc gaa gcc gcc 240

Leu Met Leu Ala Glu Asn Thr Ala Asp Glu Asp Ala Ala Glu Ala Ala

65 70 75 80 gcg cag gtg ctt tcc agc gtg gaa tct gaa atc ggg cgc acg act gac 288

Ala Gin Val Leu Ser Ser Val Glu Ser Glu Ile Gly Arg Thr Thr Asp

85 90 95 ccg gta cgc atg tac atg cgt gaa atg ggc acc gtt gaa ctg ttg acc 336

Pro Val Arg Met Tyr Met Arg Glu Met Gly Thr Val Glu Leu Leu Thr

100 105 110 cgc gaa ggc gaa att gac atc gct aag cgt att gaa gac ggg atc aac 384

Arg Glu Gly Glu Ile Asp Ile Ala Lys Arg Ile Glu Asp Gly Ile Asn

115 120 125 cag gtt caa tgc tcc gtt gct gaa tat ccg gaa gcg atc acc tat ctg 432

Gin Val Gin Cys Ser Val Ala Glu Tyr Pro Glu Ala Ile Thr Tyr Leu

130 135 140 ctg gaa cag tac gat cgt gtt gaa gca gaa gaa gcg cgt ctg tcc gat 480

Leu Glu Gin Tyr Asp Arg Val Glu Ala Glu Glu Ala Arg Leu Ser Asp

145 150 155 160 ctg atc acc ggc ttt gtt gac ccg aac gca gaa gaa gat ctg gca cct 528

Leu Ile Thr Gly Phe Val Asp Pro Asn Ala Glu Glu Asp Leu Ala Pro

165 170 175 acc gcc act cac gtc ggt tct gag ctt tcc cag gaa gat ctg gac gat 576

Thr Ala Thr His Val Gly Ser Glu Leu Ser Gin Glu Asp Leu Asp Asp

180 185 190 gac gaa gat gaa gac gaa gaa gat ggc gat gac gac agc gcc gat gat 624

Asp Glu Asp Glu Asp Glu Glu Asp Gly Asp Asp Asp Ser Ala Asp Asp

195 200 205 gac aac agc atc gac ccg gaa ctg gct cgc gaa aaa ttt gcg gaa cta 672

Asp Asn Ser Ile Asp Pro Glu Leu Ala Arg Glu Lys Phe Ala Glu Leu

210 215 220 cgc gct cag tac gtt gta acg cgt gac acc atc aaa gcg aaa ggt cgc 720

Arg Ala Gin Tyr Val Val Thr Arg Asp Thr Ile Lys Ala Lys Gly Arg

225 230 235 240 agt cac gct acc gct cag gaa gag atc ctg aaa ctg tct gaa gta ttc 768

Ser His Ala Thr Ala Gin Glu Glu Ile Leu Lys Leu Ser Glu Val Phe

245 250 255 aaa cag ttc cgc ctg gtg ccg aag cag ttt gac tac ctg gtc aac agc 816

Lys Gin Phe Arg Leu Val Pro Lys Gin Phe Asp Tyr Leu Val Asn Ser

260 265 270 atg cgc gtc atg atg gac cgc gtt cgt acg caa gaa cgt ctg atc atg 864

Met Arg Val Met Met Asp Arg Val Arg Thr Gin Glu Arg Leu Ile Met

275 280 285 aag ctc tgc gtt gag cag tgc aaa atg ccg aag aaa aac ttc att acc 912

Lys Leu Cys Val Glu Gin Cys Lys Met Pro Lys Lys Asn Phe Ile Thr

290 295 300 ctg ttt acc ggc aac gaa acc agc gat acc tgg ttc aac gcg gca att 960

Leu Phe Thr Gly Asn Glu Thr Ser Asp Thr Trp Phe Asn Ala Ala Ile 305 310 315 320 gcg atg aac aag ccg tgg tcg gaa aaa ctg cac gat gtc tct gaa gaa 1008 Ala Met Asn Lys Pro Trp Ser Glu Lys Leu His Asp Val Ser Glu Glu 325 330 335 gtg cat cgc gcc ctg caa aaa ctg cag cag att gaa gaa gaa acc ggc 1056 Val His Arg Ala Leu Gin Lys Leu Gin Gin Ile Glu Glu Glu Thr Gly

340 345 350 ctg acc atc gag cag gtt aaa gat atc aac cgt cgt atg tcc atc ggt 1104

Leu Thr Ile Glu Gin Val Lys Asp Ile Asn Arg Arg Met Ser Ile Gly

355 360 365 gaa gcg aaa gcc cgc cgt gcg aag aaa gag atg gtt gaa gcg aac tta 1152

Glu Ala Lys Ala Arg Arg Ala Lys Lys Glu Met Val Glu Ala Asn Leu

370 375 380 cgt ctg gtt att tct atc gct aag aaa tac acc aac cgt ggc ttg cag 1200

Arg Leu Val Ile Ser Ile Ala Lys Lys Tyr Thr Asn Arg Gly Leu Gin

385 390 395 400 ttc ctt gac ctg att cag gaa ggc aac atc ggt ctg atg aaa gcg gtt 1248

Phe Leu Asp Leu Ile Gin Glu Gly Asn Ile Gly Leu Met Lys Ala Val

405 410 415 gat aaa ttc gaa tac cgc cgt ggt tac aag ttc tcc acc tac gca acc 1296

Asp Lys Phe Glu Tyr Arg Arg Gly Tyr Lys Phe Ser Thr Tyr Ala Thr

420 425 430 tgg tgg atc cgt cag gcg atc acc cgc tct atc gcg gat cag gcg cgc 1344

Trp Trp Ile Arg Gin Ala Ile Thr Arg Ser Ile Ala Asp Gin Ala Arg

435 440 445 acc atc cgt att ccg gtg cat atg att gag acc atc aac aag ctc aac 1392

Thr Ile Arg Ile Pro Val His Met Ile Glu Thr Ile Asn Lys Leu Asn

450 455 460 cgt att tct cgc cag atg ctg caa gag atg ggc cgt gaa ccg acg ccg 1440

Arg Ile Ser Arg Gin Met Leu Gin Glu Met Gly Arg Glu Pro Thr Pro

465 470 475 480 gaa gaa ctg gct gaa cgt atg ctg atg ccg gaa gac aag atc cgc aaa 1488

Glu Glu Leu Ala Glu Arg Met Leu Met Pro Glu Asp Lys Ile Arg Lys

485 490 495 gtg ctg aag atc gcc aaa gag cca atc tcc atg gaa acg ccg atc ggt 1536 Val Leu Lys Ile Ala Lys Glu Pro Ile Ser Met Glu Thr Pro Ile Gly

500 505 510 gat gat gaa gat tcg cat ctg ggg gat ttc atc gag gat acc acc ctc 1584

Asp Asp Glu Asp Ser His Leu Gly Asp Phe Ile Glu Asp Thr Thr Leu

515 520 525 gag ctg ccg ctg gat tct gcg acc acc gaa agc ctg cgt gcg gca acg 1632

Glu Leu Pro Leu Asp Ser Ala Thr Thr Glu Ser Leu Arg Ala Ala Thr

530 535 540 cac gac gtg ctg gct ggc ctg acc gcg cgt gaa gca aaa gtt ctg cgt 1680

His Asp Val Leu Ala Gly Leu Thr Ala Arg Glu Ala Lys Val Leu Arg

545 550 555 560 atg cgt ttc ggt atc gat atg aac acc gac cac acg ctg gaa gaa gtg 1728 Met Arg Phe Gly Ile Asp Met Asn Thr Asp His Thr Leu Glu Glu Val 565 570 575 ggt aaa cag ttc gac gtt acc cgc gaa cgt atc cgt cag atc gaa gcg 1776

Gly Lys Gin Phe Asp Val Thr Arg Glu Arg Ile Arg Gin Ile Glu Ala 580 585 590 aag gcg ctg cgc aaa ctg cgt cac ccg agc cgt tct gaa gtg ctg cgt 1824

Lys Ala Leu Arg Lys Leu Arg His Pro Ser Arg Ser Glu Val Leu Arg 595 600 605 agc ttc ctg gac gat taa 1842 Ser Phe Leu Asp Asp

610

<210> 12

<211> 613

<212> PRT

<213> E . coli

<400> 12

Met Glu Gin Asn Pro Gin Ser Gin Leu Lys Leu Leu Val Thr Arg Gly

1 5 10 15

Lys Glu Gin Gly Tyr Leu Thr Tyr Ala Glu Val Asn Asp His Leu Pro 20 25 30

Glu Asp Ile Val Asp Ser Asp Gin Ile Glu Asp Ile Ile Gin Met Ile 35 40 45

Asn Asp Met Gly Ile Gin Val Met Glu Glu Ala Pro Asp Ala Asp Asp

50 55 60

Leu Met Leu Ala Glu Asn Thr Ala Asp Glu Asp Ala Ala Glu Ala Ala 65 70 75 80

Ala Gin Val Leu Ser Ser Val Glu Ser Glu Ile Gly Arg Thr Thr Asp 85 90 95

Pro Val Arg Met Tyr Met Arg Glu Met Gly Thr Val Glu Leu Leu Thr 100 105 110

Arg Glu Gly Glu Ile Asp Ile Ala Lys Arg Ile Glu Asp Gly Ile Asn 115 120 125

Gin Val Gin Cys Ser Val Ala Glu Tyr Pro Glu Ala Ile Thr Tyr Leu

130 135 140

Leu Glu Gin Tyr Asp Arg Val Glu Ala Glu Glu Ala Arg Leu Ser Asp 145 150 155 160 Leu Ile Thr Gly Phe Val Asp Pro Asn Ala Glu Glu Asp Leu Ala Pro 165 170 175

Thr Ala Thr His Val Gly Ser Glu Leu Ser Gin Glu Asp Leu Asp Asp 180 185 190

Asp Glu Asp Glu Asp Glu Glu Asp Gly Asp Asp Asp Ser Ala Asp Asp 195 200 205

Asp Asn Ser Ile Asp Pro Glu Leu Ala Arg Glu Lys Phe Ala Glu Leu 210 215 220

Arg Ala Gin Tyr Val Val Thr Arg Asp Thr Ile Lys Ala Lys Gly Arg 225 230 235 240

Ser His Ala Thr Ala Gin Glu Glu Ile Leu Lys Leu Ser Glu Val Phe

245 250 255

Lys Gin Phe Arg Leu Val Pro Lys Gin Phe Asp Tyr Leu Val Asn Ser 260 265 270

Met Arg Val Met Met Asp Arg Val Arg Thr Gin Glu Arg Leu Ile Met 275 280 285

Lys Leu Cys Val Glu Gin Cys Lys Met Pro Lys Lys Asn Phe Ile Thr 290 295 300

Leu Phe Thr Gly Asn Glu Thr Ser Asp Thr Trp Phe Asn Ala Ala Ile

305 310 315 320

Ala Met Asn Lys Pro Trp Ser Glu Lys Leu His Asp Val Ser Glu Glu

325 330 335 Val His Arg Ala Leu Gin Lys Leu Gin Gin Ile Glu Glu Glu Thr Gly 340 345 350

Leu Thr Ile Glu Gin Val Lys Asp Ile Asn Arg Arg Met Ser Ile Gly 355 360 365

Glu Ala Lys Ala Arg Arg Ala Lys Lys Glu Met Val Glu Ala Asn Leu 370 375 380

Arg Leu Val Ile Ser Ile Ala Lys Lys Tyr Thr Asn Arg Gly Leu Gin 385 390 395 400

Phe Leu Asp Leu Ile Gin Glu Gly Asn Ile Gly Leu Met Lys Ala Val

405 410 415 Asp Lys Phe Glu Tyr Arg Arg Gly Tyr Lys Phe Ser Thr Tyr Ala Thr

420 425 430

Trp Trp Ile Arg Gin Ala Ile Thr Arg Ser Ile Ala Asp Gin Ala Arg

435 440 445

Thr Ile Arg Ile Pro Val His Met Ile Glu Thr Ile Asn Lys Leu Asn

450 455 460

Arg Ile Ser Arg Gin Met Leu Gin Glu Met Gly Arg Glu Pro Thr Pro 465 470 475 480

Glu Glu Leu Ala Glu Arg Met Leu Met Pro Glu Asp Lys Ile Arg Lys 485 490 495 Val Leu Lys Ile Ala Lys Glu Pro Ile Ser Met Glu Thr Pro Ile Gly

500 505 510

Asp Asp Glu Asp Ser His Leu Gly Asp Phe Ile Glu Asp Thr Thr Leu 515 520 525

Glu Leu Pro Leu Asp Ser Ala Thr Thr Glu Ser Leu Arg Ala Ala Thr 530 535 540

His Asp Val Leu Ala Gly Leu Thr Ala Arg Glu Ala Lys Val Leu Arg 545 550 555 560

Met Arg Phe Gly Ile Asp Met Asn Thr Asp His Thr Leu Glu Glu Val 565 570 575

Gly Lys Gin Phe Asp Val Thr Arg Glu Arg Ile Arg Gin Ile Glu Ala

580 585 590

Lys Ala Leu Arg Lys Leu Arg His Pro Ser Arg Ser Glu Val Leu Arg 595 600 605

Ser Phe Leu Asp Asp 610

<210> 13

<211> 546

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (546)

<223> E . coli essential gene nusG <400> 13 atg tct gaa gct cct aaa aag cgc tgg tac gtc gtt cag gcg ttt tcc 48

Met Ser Glu Ala Pro Lys Lys Arg Trp Tyr Val Val Gin Ala Phe Ser

1 5 10 15 ggt ttt gaa ggc cgc gta gca acg tcg ctg cgt gag cat atc aaa tta 96

Gly Phe Glu Gly Arg Val Ala Thr Ser Leu Arg Glu His Ile Lys Leu

20 25 30 cac aac atg gaa gat ttg ttt ggt gaa gtc atg gta cca acc gaa gaa 144 His Asn Met Glu Asp Leu Phe Gly Glu Val Met Val Pro Thr Glu Glu 35 40 45 gtg gtt gaa atc cgt ggc ggt cag cgt cgc aaa agc gaa cgt aaa ttc 192 Val Val Glu Ile Arg Gly Gly Gin Arg Arg Lys Ser Glu Arg Lys Phe 50 55 60 ttc cct ggc tac gtc ctc gtt cag atg gtg atg aac gac gcg agc tgg 240

Phe Pro Gly Tyr Val Leu Val Gin Met Val Met Asn Asp Ala Ser Trp

65 70 75 80 cac ctg gtg cgc agc gta ccg cgt gtg atg ggc ttc atc ggc ggt act 288

His Leu Val Arg Ser Val Pro Arg Val Met Gly Phe Ile Gly Gly Thr

85 90 95 tcc gat cgt cct gcg cca atc agc gat aaa gaa gtc gat gcg att atg 336

Ser Asp Arg Pro Ala Pro Ile Ser Asp Lys Glu Val Asp Ala Ile Met

100 105 110 aac cgc ctg cag cag gtt ggt gat aag ccg cgt ccg aaa acg ctg ttt 384

Asn Arg Leu Gin Gin Val Gly Asp Lys Pro Arg Pro Lys Thr Leu Phe

115 120 125 gaa ccg ggt gaa atg gtc cgt gtt aat gat ggt ccg ttc gct gac ttc 432

Glu Pro Gly Glu Met Val Arg Val Asn Asp Gly Pro Phe Ala Asp Phe

130 135 140 aac ggt gtt gtt gaa gaa gtg gat tac gag aaa tct cgt ctg aaa gtg 480

Asn Gly Val Val Glu Glu Val Asp Tyr Glu Lys Ser Arg Leu Lys Val

145 150 155 160 tct gtt tct atc ttc ggt cgt gcg acc ccg gta gag ctg gac ttc agc 528

Ser Val Ser Ile Phe Gly Arg Ala Thr Pro Val Glu Leu Asp Phe Ser

165 170 175 cag gtt gaa aaa gcc taa 546

Gin Val Glu Lys Ala

180

<210> 14

<211> 181

<212> PRT

<213> E . coli

<400> 14

Met Ser Glu Ala Pro Lys Lys Arg Trp Tyr Val Val Gin Ala Phe Ser

1 5 10 15

Gly Phe Glu Gly Arg Val Ala Thr Ser Leu Arg Glu His Ile Lys Leu 20 25 30

His Asn Met Glu Asp Leu Phe Gly Glu Val Met Val Pro Thr Glu Glu 35 40 45 Val Val Glu Ile Arg Gly Gly Gin Arg Arg Lys Ser Glu Arg Lys Phe 50 55 60

Phe Pro Gly Tyr Val Leu Val Gin Met Val Met Asn Asp Ala Ser Trp 65 70 75 80

His Leu Val Arg Ser Val Pro Arg Val Met Gly Phe Ile Gly Gly Thr

85 90 95

Ser Asp Arg Pro Ala Pro Ile Ser Asp Lys Glu Val Asp Ala Ile Met 100 105 110

Asn Arg Leu Gin Gin Val Gly Asp Lys Pro Arg Pro Lys Thr Leu Phe 115 120 125

Glu Pro Gly Glu Met Val Arg Val Asn Asp Gly Pro Phe Ala Asp Phe

130 135 140

Asn Gly Val Val Glu Glu Val Asp Tyr Glu Lys Ser Arg Leu Lys Val 145 150 155 160

Ser Val Ser Ile Phe Gly Arg Ala Thr Pro Val Glu Leu Asp Phe Ser

165 170 175

Gin Val Glu Lys Ala 180

<210> 15

<211> 216

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (216)

<223> E . coli essential gene rpsU

<400> 15 atg ccg gta att aaa gta cgt gaa aac gag ccg ttc gac gta gct ctg 48

Met Pro Val Ile Lys Val Arg Glu Asn Glu Pro Phe Asp Val Ala Leu

1 5 10 15 cgt cgc ttc aag cgt tcc tgc gaa aaa gca ggt gtt ctg gcg gaa gtt 96

Arg Arg Phe Lys Arg Ser Cys Glu Lys Ala Gly Val Leu Ala Glu Val

20 25 30 cgt cgt cgt gag ttc tat gaa aaa ccg act acc gaa cgt aag cgc gct 144

Arg Arg Arg Glu Phe Tyr Glu Lys Pro Thr Thr Glu Arg Lys Arg Ala

35 40 45 aaa gct tct gca gtg aaa cgt cac gcg aag aaa ctg gct cgc gaa aac 192

Lys Ala Ser Ala Val Lys Arg His Ala Lys Lys Leu Ala Arg Glu Asn

50 55 60 gca cgc cgc act cgt ctg tac taa 216

Ala Arg Arg Thr Arg Leu Tyr

65 70

<210> 16

<211> 71

<212> PRT

<213> E . coli

<400> 16

Met Pro Val Ile Lys Val Arg Glu Asn Glu Pro Phe Asp Val Ala Leu

1 5 10 15

Arg Arg Phe Lys Arg Ser Cys Glu Lys Ala Gly Val Leu Ala Glu Val 20 25 30

Arg Arg Arg Glu Phe Tyr Glu Lys Pro Thr Thr Glu Arg Lys Arg Ala 35 40 45

Lys Ala Ser Ala Val Lys Arg His Ala Lys Lys Leu Ala Arg Glu Asn 50 55 60

Ala Arg Arg Thr Arg Leu Tyr 65 70

<210> 17

<211> 216

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (216)

<223> E . coli essential gene accD

<400> 17 atg ccg gta att aaa gta cgt gaa aac gag ccg ttc gac gta gct ctg 48 Met Pro Val Ile Lys Val Arg Glu Asn Glu Pro Phe Asp Val Ala Leu 1 5 10 15 cgt cgc ttc aag cgt tec tgc gaa aaa gca ggt gtt ctg gcg gaa gtt 96 Arg Arg Phe Lys Arg Ser Cys Glu Lys Ala Gly Val Leu Ala Glu Val 20 25 30 cgt cgt cgt gag ttc tat gaa aaa ccg act acc gaa cgt aag cgc gct 144 Arg Arg Arg Glu Phe Tyr Glu Lys Pro Thr Thr Glu Arg Lys Arg Ala

35 40 45 aaa gct tct gca gtg aaa cgt cac gcg aag aaa ctg gct cgc gaa aac 192 Lys Ala Ser Ala Val Lys Arg His Ala Lys Lys Leu Ala Arg Glu Asn 50 55 60 gca cgc cgc act cgt ctg tac taa 216 Ala Arg Arg Thr Arg Leu Tyr 65 70

<210> 18

<211> 71

<212> PRT

<213> E . coli

<400> 18

Met Pro Val Ile Lys Val Arg Glu Asn Glu Pro Phe Asp Val Ala Leu 1 5 10 15

Arg Arg Phe Lys Arg Ser Cys Glu Lys Ala Gly Val Leu Ala Glu Val 20 25 30

Arg Arg Arg Glu Phe Tyr Glu Lys Pro Thr Thr Glu Arg Lys Arg Ala 35 40 45

Lys Ala Ser Ala Val Lys Arg His Ala Lys Lys Leu Ala Arg Glu Asn 50 55 60

Ala Arg Arg Thr Arg Leu Tyr 65 70

<210> 19

<211> 216

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (216)

<223> E coli essential gene degS

<400> 19 atg ccg gta att aaa gta cgt gaa aac gag ccg ttc gac gta gct ctg 48

Met Pro Val Ile Lys Val Arg Glu Asn Glu Pro Phe Asp Val Ala Leu

1 5 10 15 cgt cgc ttc aag cgt tcc tgc gaa aaa gca ggt gtt ctg gcg gaa gtt 96

Arg Arg Phe Lys Arg Ser Cys Glu Lys Ala Gly Val Leu Ala Glu Val

20 25 30 cgt cgt cgt gag ttc tat gaa aaa ccg act acc gaa cgt aag cgc gct 144

Arg Arg Arg Glu Phe Tyr Glu Lys Pro Thr Thr Glu Arg Lys Arg Ala

35 40 45 aaa gct tct gca gtg aaa cgt cac gcg aag aaa ctg gct cgc gaa aac 192

Lys Ala Ser Ala Val Lys Arg His Ala Lys Lys Leu Ala Arg Glu Asn 50 55 60 gca cgc cgc act cgt ctg tac taa 216 Ala Arg Arg Thr Arg Leu Tyr 65 70

<210> 20

<211> 71

<212> PRT

<213> E . coll

<400> 20

Met Pro Val Ile Lys Val Arg Glu Asn Glu Pro Phe Asp Val Ala Leu 1 5 10 15

Arg Arg Phe Lys Arg Ser Cys Glu Lys Ala Gly Val Leu Ala Glu Val 20 25 30

Arg Arg Arg Glu Phe Tyr Glu Lys Pro Thr Thr Glu Arg Lys Arg Ala 35 40 45

Lys Ala Ser Ala Val Lys Arg His Ala Lys Lys Leu Ala Arg Glu Asn 50 55 60

Ala Arg Arg Thr Arg Leu Tyr 65 70

<210> 21

<211> 531

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (531)

<223> E . coll essential gene fldA

<400> 21 atg gct atc act ggc atc ttt ttc ggc agc gac acc ggt aat acc gaa 48

Met Ala Ile Thr Gly Ile Phe Phe Gly Ser Asp Thr Gly Asn Thr Glu

1 5 10 15 aat atc gca aaa atg att caa aaa cag ctt ggt aaa gac gtt gcc gat 96

Asn Ile Ala Lys Met Ile Gin Lys Gin Leu Gly Lys Asp Val Ala Asp

20 25 30 gtc cat gac att gca aaa agc agc aaa gaa gat ctg gaa gct tat gac 144 Val His Asp Ile Ala Lys Ser Ser Lys Glu Asp Leu Glu Ala Tyr Asp

35 40 45 att ctg ctg ctg ggc atc cca acc tgg tat tac ggc gaa gcg cag tgt 192 Ile Leu Leu Leu Gly Ile Pro Thr Trp Tyr Tyr Gly Glu Ala Gin Cys

50 55 60 gac tgg gat gac ttc ttc ccg act ctc gaa gag att gat ttc aat ggc 240 Asp Trp Asp Asp Phe Phe Pro Thr Leu Glu Glu Ile Asp Phe Asn Gly 65 70 75 80 aaa ctg gtt gcg ctg ttt ggt tgt ggt gac cag gaa gat tac gcc gaa 288

Lys Leu Val Ala Leu Phe Gly Cys Gly Asp Gin Glu Asp Tyr Ala Glu 85 90 95 tat ttc tgc gac gca ttg ggc acc atc cgc gac atc att gaa ccg cgc 336

Tyr Phe Cys Asp Ala Leu Gly Thr Ile Arg Asp Ile Ile Glu Pro Arg 100 105 110 ggt gca acc atc gtt ggt cac tgg cca act gcg ggc tat cat ttc gaa 384

Gly Ala Thr Ile Val Gly His Trp Pro Thr Ala Gly Tyr His Phe Glu 115 120 125 gca tca aaa ggt ctg gca gat gac gac cac ttt gtc ggt ctg gct atc 432

Ala Ser Lys Gly Leu Ala Asp Asp Asp His Phe Val Gly Leu Ala Ile 130 135 140 gac gaa gac cgt cag ccg gaa ctg acc gct gaa cgt gta gaa aaa tgg 480 Asp Glu Asp Arg Gin Pro Glu Leu Thr Ala Glu Arg Val Glu Lys Trp 145 150 155 160 gtt aaa cag att tct gaa gag ttg cat ctc gac gaa att ctc aat gcc 528 Val Lys Gin Ile Ser Glu Glu Leu His Leu Asp Glu Ile Leu Asn Ala 165 170 175 tga 531

<210> 22

<211> 176

<212> PRT

<213> E . coll

<400> 22

Met Ala Ile Thr Gly Ile Phe Phe Gly Ser Asp Thr Gly Asn Thr Glu

1 5 10 15

Asn Ile Ala Lys Met Ile Gin Lys Gin Leu Gly Lys Asp Val Ala Asp 20 25 30 Val His Asp Ile Ala Lys Ser Ser Lys Glu Asp Leu Glu Ala Tyr Asp 35 40 45 Ile Leu Leu Leu Gly Ile Pro Thr Trp Tyr Tyr Gly Glu Ala Gin Cys 50 55 60

Asp Trp Asp Asp Phe Phe Pro Thr Leu Glu Glu Ile Asp Phe Asn Gly 65 70 75 80

Lys Leu Val Ala Leu Phe Gly Cys Gly Asp Gin Glu Asp Tyr Ala Glu 85 90 95

Tyr Phe Cys Asp Ala Leu Gly Thr Ile Arg Asp Ile Ile Glu Pro Arg 100 105 110 Gly Ala Thr Ile Val Gly His Trp Pro Thr Ala Gly Tyr His Phe Glu 115 120 125

Ala Ser Lys Gly Leu Ala Asp Asp Asp His Phe Val Gly Leu Ala Ile

130 135 140

Asp Glu Asp Arg Gin Pro Glu Leu Thr Ala Glu Arg Val Glu Lys Trp 145 150 155 160 Val Lys Gin Ile Ser Glu Glu Leu His Leu Asp Glu Ile Leu Asn Ala

165 170 175

<210> 23

<211> 960

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . ( 960)

<223> E . coli essential gene ftsN

<400> 23 gtg gca caa cga gat tat gta cgc cgc agc caa ccg gca cct tcg egg 48 Val Ala Gin Arg Asp Tyr Val Arg Arg Ser Gin Pro Ala Pro Ser Arg 1 5 10 15 cga aaa aag agc acc tca egg aaa aag caa cga aat ctg cct gcg gtt 96

Arg Lys Lys Ser Thr Ser Arg Lys Lys Gin Arg Asn Leu Pro Ala Val

20 25 30 tct ccc gct atg gtc gct att gct gcc gcc gtt ctt gtg acc ttt atc 144

Ser Pro Ala Met Val Ala Ile Ala Ala Ala Val Leu Val Thr Phe Ile

35 40 45 ggt ggt ctg tac ttc att acg cat cac aag aaa gaa gag tcc gag acg 192

Gly Gly Leu Tyr Phe Ile Thr His His Lys Lys Glu Glu Ser Glu Thr

50 55 60 ctg caa agc cag aaa gtg acc gga aac gga cta cca cca aaa cca gaa 240

Leu Gin Ser Gin Lys Val Thr Gly Asn Gly Leu Pro Pro Lys Pro Glu

65 70 75 80 gaa cgc tgg cgc tac att aaa gag ctg gaa agt cgc cag ccg gga gtg 288

Glu Arg Trp Arg Tyr Ile Lys Glu Leu Glu Ser Arg Gin Pro Gly Val

85 90 95 cgt gcg ccc aca gaa cct tct gcc ggt ggt gaa gtg aaa acg ccg gag 336

Arg Ala Pro Thr Glu Pro Ser Ala Gly Gly Glu Val Lys Thr Pro Glu

100 105 110 caa ctg aca cca gaa caa cgt cag ctt ctt gaa caa atg cag gct gat 384

Gin Leu Thr Pro Glu Gin Arg Gin Leu Leu Glu Gin Met Gin Ala Asp

115 120 125 atg cgc cag cag cca acg cag ctg gtt gaa gtt ccg tgg aat gaa cag 432 Met Arg Gin Gin Pro Thr Gin Leu Val Glu Val Pro Trp Asn Glu Gin

130 135 140 acg ccg gaa cag cgt cag caa acg cta cag cgc caa cgt cag gcg cag 480

Thr Pro Glu Gin Arg Gin Gin Thr Leu Gin Arg Gin Arg Gin Ala Gin

145 150 155 160 caa tta gcg gaa cag caa cgc ctg gct cag cag tcg cgt acg act gaa 528

Gin Leu Ala Glu Gin Gin Arg Leu Ala Gin Gin Ser Arg Thr Thr Glu

165 170 175 caa agc tgg cag cag cag acg cgt acg tcg caa gcc gca ccg gta cag 576

Gin Ser Trp Gin Gin Gin Thr Arg Thr Ser Gin Ala Ala Pro Val Gin

180 185 190 gcg caa ccg cgc cag tct aaa ccg gct tct tct cag cag ccg tac cag 624

Ala Gin Pro Arg Gin Ser Lys Pro Ala Ser Ser Gin Gin Pro Tyr Gin

195 200 205 gat ctg ctg caa act cct gcg cac acg act gcg cag tcg aag ccg cag 672

Asp Leu Leu Gin Thr Pro Ala His Thr Thr Ala Gin Ser Lys Pro Gin

210 215 220 caa gct gcg cca gtt gct cgt gcc gct gac gcg cca aaa ccg acg gcg 720

Gin Ala Ala Pro Val Ala Arg Ala Ala Asp Ala Pro Lys Pro Thr Ala

225 230 235 240 gag aaa aaa gac gaa cgc cgc tgg atg gtg cag tgc ggt tcg ttc aga 768

Glu Lys Lys Asp Glu Arg Arg Trp Met Val Gin Cys Gly Ser Phe Arg

245 250 255 ggc gcg gaa cag gca gag acg gta cgt gct cag ctg gcg ttc gaa ggc 816

Gly Ala Glu Gin Ala Glu Thr Val Arg Ala Gin Leu Ala Phe Glu Gly

260 265 270 ttt gac tcg aaa atc acc acc aac aat ggc tgg aat cgt gtg gtc att 864

Phe Asp Ser Lys Ile Thr Thr Asn Asn Gly Trp Asn Arg Val Val Ile

275 280 285 ggc ccg gtg aaa ggc aaa gag aac gca gac agc acc ctc aat egg ttg 912

Gly Pro Val Lys Gly Lys Glu Asn Ala Asp Ser Thr Leu Asn Arg Leu

290 295 300 aag atg gcg ggc cat aca aac tgc att egg ctc gcc gcc ggg ggt tga 960

Lys Met Ala Gly His Thr Asn Cys Ile Arg Leu Ala Ala Gly Gly

305 310 315

<210> 24

<211> 319

<212> PRT

<213> E . coll

<400> 24 Val Ala Gin Arg Asp Tyr Val Arg Arg Ser Gin Pro Ala Pro Ser Arg

1 5 10 15

Arg Lys Lys Ser Thr Ser Arg Lys Lys Gin Arg Asn Leu Pro Ala Val 20 25 30 Ser Pro Ala Met Val Ala Ile Ala Ala Ala Val Leu Val Thr Phe Ile 35 40 45

Gly Gly Leu Tyr Phe Ile Thr His His Lys Lys Glu Glu Ser Glu Thr 50 55 60

Leu Gin Ser Gin Lys Val Thr Gly Asn Gly Leu Pro Pro Lys Pro Glu 65 70 75 80

Glu Arg Trp Arg Tyr Ile Lys Glu Leu Glu Ser Arg Gin Pro Gly Val 85 90 95

Arg Ala Pro Thr Glu Pro Ser Ala Gly Gly Glu Val Lys Thr Pro Glu 100 105 110

Gin Leu Thr Pro Glu Gin Arg Gin Leu Leu Glu Gin Met Gin Ala Asp 115 120 125

Met Arg Gin Gin Pro Thr Gin Leu Val Glu Val Pro Trp Asn Glu Gin 130 135 140

Thr Pro Glu Gin Arg Gin Gin Thr Leu Gin Arg Gin Arg Gin Ala Gin

145 150 155 160

Gin Leu Ala Glu Gin Gin Arg Leu Ala Gin Gin Ser Arg Thr Thr Glu 165 170 175

Gin Ser Trp Gin Gin Gin Thr Arg Thr Ser Gin Ala Ala Pro Val Gin 180 185 190

Ala Gin Pro Arg Gin Ser Lys Pro Ala Ser Ser Gin Gin Pro Tyr Gin 195 200 205

Asp Leu Leu Gin Thr Pro Ala His Thr Thr Ala Gin Ser Lys Pro Gin 210 215 220

Gin Ala Ala Pro Val Ala Arg Ala Ala Asp Ala Pro Lys Pro Thr Ala

225 230 235 240

Glu Lys Lys Asp Glu Arg Arg Trp Met Val Gin Cys Gly Ser Phe Arg

245 250 255

Gly Ala Glu Gin Ala Glu Thr Val Arg Ala Gin Leu Ala Phe Glu Gly 260 265 270

Phe Asp Ser Lys Ile Thr Thr Asn Asn Gly Trp Asn Arg Val Val Ile 275 280 285 Gly Pro Val Lys Gly Lys Glu Asn Ala Asp Ser Thr Leu Asn Arg Leu 290 295 300

Lys Met Ala Gly His Thr Asn Cys Ile Arg Leu Ala Ala Gly Gly 305 310 315

<210> 25

<211> 1935

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1935)

<223> E . coll essential gene hflB

<400> 25 atg gcg aaa aac cta ata ctc tgg ctg gtc att gcc gtt gtg ctg atg 48

Met Ala Lys Asn Leu Ile Leu Trp Leu Val Ile Ala Val Val Leu Met

1 5 10 15 tca gta ttc cag agc ttt ggg ccc agc gag tct aat ggc cgt aag gtg 96

Ser Val Phe Gin Ser Phe Gly Pro Ser Glu Ser Asn Gly Arg Lys Val

20 25 30 gat tac tct acc ttc cta caa gag gtc aat aac gac cag gtt cgt gaa 144

Asp Tyr Ser Thr Phe Leu Gin Glu Val Asn Asn Asp Gin Val Arg Glu

35 40 45 gcg cgt atc aac gga cgt gaa atc aac gtt acc aag aaa gat agt aac 192

Ala Arg Ile Asn Gly Arg Glu Ile Asn Val Thr Lys Lys Asp Ser Asn

50 55 60 cgt tat acc act tac att ccg gtt cag gat ccg aaa tta ctg gat aac 240

Arg Tyr Thr Thr Tyr Ile Pro Val Gin Asp Pro Lys Leu Leu Asp Asn

65 70 75 80 ctg ttg acc aag aac gtc aag gtt gtc ggt gaa ccg cct gaa gaa cca 288

Leu Leu Thr Lys Asn Val Lys Val Val Gly Glu Pro Pro Glu Glu Pro

85 90 95 agc ctg ctg gct tct atc ttc atc tec tgg ttc ccg atg ctg ttg ctg 336

Ser Leu Leu Ala Ser Ile Phe Ile Ser Trp Phe Pro Met Leu Leu Leu

100 105 110 att ggt gtc tgg atc ttc ttc atg cgt caa atg cag ggc ggc ggt ggc 384 Ile Gly Val Trp Ile Phe Phe Met Arg Gin Met Gin Gly Gly Gly Gly

115 120 125 aaa ggt gcc atg tcg ttt ggt aag agc aaa gcg cgc atg ctg acg gaa 432

Lys Gly Ala Met Ser Phe Gly Lys Ser Lys Ala Arg Met Leu Thr Glu

130 135 140 gat cag atc aaa acg acc ttt gct gac gtt gcg ggc tgc gac gaa gca 480

Asp Gin Ile Lys Thr Thr Phe Ala Asp Val Ala Gly Cys Asp Glu Ala

145 150 155 160 aaa gaa gaa gtt gct gaa ctg gtt gag tat ctg cgc gag ccg agc cgc 528

Lys Glu Glu Val Ala Glu Leu Val Glu Tyr Leu Arg Glu Pro Ser Arg 165 170 175 ttc cag aaa ctc ggc ggt aag atc ccg aaa ggc gtc ttg atg gtc ggt 576

Phe Gin Lys Leu Gly Gly Lys Ile Pro Lys Gly Val Leu Met Val Gly

180 185 190 cct ccg ggt acc ggt aaa acg ctg ctg gcg aaa gcg att gca ggc gaa 624

Pro Pro Gly Thr Gly Lys Thr Leu Leu Ala Lys Ala Ile Ala Gly Glu

195 200 205 gcg aaa gtt ccg ttc ttt act atc tcc ggt tct gac ttc gta gaa atg 672

Ala Lys Val Pro Phe Phe Thr Ile Ser Gly Ser Asp Phe Val Glu Met

210 215 220 ttc gtc ggt gtg ggt gca tcc cgt gtt cgt gac atg ttc gaa cag gcg 720

Phe Val Gly Val Gly Ala Ser Arg Val Arg Asp Met Phe Glu Gin Ala

225 230 235 240 aag aaa gcg gca ccg tgc atc atc ttt atc gat gaa atc gac gcc gta 768

Lys Lys Ala Ala Pro Cys Ile Ile Phe Ile Asp Glu Ile Asp Ala Val

245 250 255 ggc cgc cag cgt ggc gct ggt ctg ggc ggt ggt cac gat gaa cgt gaa 816

Gly Arg Gin Arg Gly Ala Gly Leu Gly Gly Gly His Asp Glu Arg Glu

260 265 270 cag act ctg aac cag atg ctg gtt gag atg gat ggc ttc gaa ggt aac 864

Gin Thr Leu Asn Gin Met Leu Val Glu Met Asp Gly Phe Glu Gly Asn

275 280 285 gaa ggt atc atc gtt atc gcc gcg act aac cgt ccg gac gtt ctc gac 912

Glu Gly Ile Ile Val Ile Ala Ala Thr Asn Arg Pro Asp Val Leu Asp

290 295 300 ccg gcc ctg ctg cgt cct ggc cgt ttc gac cgt cag gtt gtg gtc ggc 960

Pro Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg Gin Val Val Val Gly

305 310 315 320 ttg cca gat gtt cgc ggt cgt gag cag atc ctg aaa gtt cac atg cgt 1008

Leu Pro Asp Val Arg Gly Arg Glu Gin Ile Leu Lys Val His Met Arg

325 330 335 cgc gta cca ttg gca ccc gat atc gac gcg gca atc att gcc cgt ggt 1056

Arg Val Pro Leu Ala Pro Asp Ile Asp Ala Ala Ile Ile Ala Arg Gly

340 345 350 act cct ggt ttc tcc ggt gct gac ctg gcg aac ctg gtg aac gaa gcg 1104

Thr Pro Gly Phe Ser Gly Ala Asp Leu Ala Asn Leu Val Asn Glu Ala

355 360 365 gca ctg ttc gct gct cgt ggc aac aaa cgc gtt gtg tcg atg gtt gag 1152

Ala Leu Phe Ala Ala Arg Gly Asn Lys Arg Val Val Ser Met Val Glu

370 375 380 ttc gag aaa gcg aaa gac aaa atc atg atg ggt gcg gaa cgt cgc tcc 1200

Phe Glu Lys Ala Lys Asp Lys Ile Met Met Gly Ala Glu Arg Arg Ser

385 390 395 400 atg gtg atg acg gaa gcg cag aaa gaa tcg acg gct tac cac gaa gcg 1248

Met Val Met Thr Glu Ala Gin Lys Glu Ser Thr Ala Tyr His Glu Ala

405 410 415 ggt cat gcg att atc ggt cgc ctg gtg ccg gaa cac gat ccg gtg cac 1296 Gly His Ala Ile Ile Gly Arg Leu Val Pro Glu His Asp Pro Val His

420 425 430 aaa gtg acg att atc cca cgc ggt cgt gcg ctg ggt gtg act ttc ttc 1344

Lys Val Thr Ile Ile Pro Arg Gly Arg Ala Leu Gly Val Thr Phe Phe

435 440 445 ttg cct gag ggc gac gca atc agc gcc agc cgt cag aaa ctg gaa agc 1392

Leu Pro Glu Gly Asp Ala Ile Ser Ala Ser Arg Gin Lys Leu Glu Ser

450 455 460 cag att tct acg ctg tac ggt ggt cgt ctg gca gaa gag atc atc tac 1440

Gin Ile Ser Thr Leu Tyr Gly Gly Arg Leu Ala Glu Glu Ile Ile Tyr

465 470 475 480 ggg ccg gaa cat gta tct acc ggt gcg tcc aac gat att aaa gtt gcg 1488

Gly Pro Glu His Val Ser Thr Gly Ala Ser Asn Asp Ile Lys Val Ala

485 490 495 acc aac ctg gca cgt aac atg gtg act cag tgg ggc ttc tct gag aaa 1536

Thr Asn Leu Ala Arg Asn Met Val Thr Gin Trp Gly Phe Ser Glu Lys

500 505 510 ttg ggt cca ctg ctg tac gcg gaa gaa gaa ggt gaa gtg ttc ctc ggc 1584

Leu Gly Pro Leu Leu Tyr Ala Glu Glu Glu Gly Glu Val Phe Leu Gly

515 520 525 cgt agc gta gcg aaa gcg aaa cat atg tcc gat gaa act gca cgt atc 1632

Arg Ser Val Ala Lys Ala Lys His Met Ser Asp Glu Thr Ala Arg Ile

530 535 540 atc gac cag gaa gtg aaa gca ctg att gag cgt aac tat aat cgt gcg 1680 Ile Asp Gin Glu Val Lys Ala Leu Ile Glu Arg Asn Tyr Asn Arg Ala

545 550 555 560 cgt cag ctt ctg acc gac aat atg gat att ctg cat gcg atg aaa gat 1728

Arg Gin Leu Leu Thr Asp Asn Met Asp Ile Leu His Ala Met Lys Asp

565 570 575 gct ctc atg aaa tat gag act atc gac gca ccg cag att gat gac ctg 1776

Ala Leu Met Lys Tyr Glu Thr Ile Asp Ala Pro Gin Ile Asp Asp Leu

580 585 590 atg gca cgt cgc gat gta cgt ccg cca gcg ggc tgg gaa gaa cca ggc 1824

Met Ala Arg Arg Asp Val Arg Pro Pro Ala Gly Trp Glu Glu Pro Gly

595 600 605 gct tct aac aat tct ggc gac aat ggt agt cca aag gct cct cgt ccg 1872

Ala Ser Asn Asn Ser Gly Asp Asn Gly Ser Pro Lys Ala Pro Arg Pro

610 615 620 gtt gat gaa ccg cgt acg ccg aac ccg ggt aac acc atg tca gag cag 1920 Val Asp Glu Pro Arg Thr Pro Asn Pro Gly Asn Thr Met Ser Glu Gin

625 630 635 640 tta ggc gac aag taa 1935

Leu Gly Asp Lys

<210> 26

<211> 644

<212> PRT <213> E . coli

<400> 26

Met Ala Lys Asn Leu Ile Leu Trp Leu Val Ile Ala Val Val Leu Met

1 5 10 15

Ser Val Phe Gin Ser Phe Gly Pro Ser Glu Ser Asn Gly Arg Lys Val 20 25 30

Asp Tyr Ser Thr Phe Leu Gin Glu Val Asn Asn Asp Gin Val Arg Glu 35 40 45

Ala Arg Ile Asn Gly Arg Glu Ile Asn Val Thr Lys Lys Asp Ser Asn 50 55 60

Arg Tyr Thr Thr Tyr Ile Pro Val Gin Asp Pro Lys Leu Leu Asp Asn 65 70 75 80

Leu Leu Thr Lys Asn Val Lys Val Val Gly Glu Pro Pro Glu Glu Pro

85 90 95

Ser Leu Leu Ala Ser Ile Phe Ile Ser Trp Phe Pro Met Leu Leu Leu

100 105 110 Ile Gly Val Trp Ile Phe Phe Met Arg Gin Met Gin Gly Gly Gly Gly

115 120 125

Lys Gly Ala Met Ser Phe Gly Lys Ser Lys Ala Arg Met Leu Thr Glu

130 135 140

Asp Gin Ile Lys Thr Thr Phe Ala Asp Val Ala Gly Cys Asp Glu Ala 145 150 155 160

Lys Glu Glu Val Ala Glu Leu Val Glu Tyr Leu Arg Glu Pro Ser Arg

165 170 175

Phe Gin Lys Leu Gly Gly Lys Ile Pro Lys Gly Val Leu Met Val Gly

180 185 190

Pro Pro Gly Thr Gly Lys Thr Leu Leu Ala Lys Ala Ile Ala Gly Glu 195 200 205

Ala Lys Val Pro Phe Phe Thr Ile Ser Gly Ser Asp Phe Val Glu Met

210 215 220

Phe Val Gly Val Gly Ala Ser Arg Val Arg Asp Met Phe Glu Gin Ala 225 230 235 240 Lys Lys Ala Ala Pro Cys Ile Ile Phe Ile Asp Glu Ile Asp Ala Val 245 250 255

Gly Arg Gin Arg Gly Ala Gly Leu Gly Gly Gly His Asp Glu Arg Glu 260 265 270

Gin Thr Leu Asn Gin Met Leu Val Glu Met Asp Gly Phe Glu Gly Asn

275 280 285

Glu Gly Ile Ile Val Ile Ala Ala Thr Asn Arg Pro Asp Val Leu Asp

290 295 300

Pro Ala Leu Leu Arg Pro Gly Arg Phe Asp Arg Gin Val Val Val Gly 305 310 315 320

Leu Pro Asp Val Arg Gly Arg Glu Gin Ile Leu Lys Val His Met Arg 325 330 335

Arg Val Pro Leu Ala Pro Asp Ile Asp Ala Ala Ile Ile Ala Arg Gly 340 345 350

Thr Pro Gly Phe Ser Gly Ala Asp Leu Ala Asn Leu Val Asn Glu Ala

355 360 365

Ala Leu Phe Ala Ala Arg Gly Asn Lys Arg Val Val Ser Met Val Glu 370 375 380

Phe Glu Lys Ala Lys Asp Lys Ile Met Met Gly Ala Glu Arg Arg Ser 385 390 395 400

Met Val Met Thr Glu Ala Gin Lys Glu Ser Thr Ala Tyr His Glu Ala 405 410 415

Gly His Ala Ile Ile Gly Arg Leu Val Pro Glu His Asp Pro Val His 420 425 430

Lys Val Thr Ile Ile Pro Arg Gly Arg Ala Leu Gly Val Thr Phe Phe 435 440 445

Leu Pro Glu Gly Asp Ala Ile Ser Ala Ser Arg Gin Lys Leu Glu Ser 450 455 460

Gin Ile Ser Thr Leu Tyr Gly Gly Arg Leu Ala Glu Glu Ile Ile Tyr 465 470 475 480

Gly Pro Glu His Val Ser Thr Gly Ala Ser Asn Asp Ile Lys Val Ala 485 490 495 Thr Asn Leu Ala Arg Asn Met Val Thr Gin Trp Gly Phe Ser Glu Lys 500 505 510

Leu Gly Pro Leu Leu Tyr Ala Glu Glu Glu Gly Glu Val Phe Leu Gly 515 520 525

Arg Ser Val Ala Lys Ala Lys His Met Ser Asp Glu Thr Ala Arg Ile

530 535 540 Ile Asp Gin Glu Val Lys Ala Leu Ile Glu Arg Asn Tyr Asn Arg Ala

545 550 555 560

Arg Gin Leu Leu Thr Asp Asn Met Asp Ile Leu His Ala Met Lys Asp 565 570 575

Ala Leu Met Lys Tyr Glu Thr Ile Asp Ala Pro Gin Ile Asp Asp Leu 580 585 590

Met Ala Arg Arg Asp Val Arg Pro Pro Ala Gly Trp Glu Glu Pro Gly 595 600 605

Ala Ser Asn Asn Ser Gly Asp Asn Gly Ser Pro Lys Ala Pro Arg Pro

610 615 620 Val Asp Glu Pro Arg Thr Pro Asn Pro Gly Asn Thr Met Ser Glu Gin 625 630 635 640

Leu Gly Asp Lys

<210> 27

<211> 612

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . ( 612 )

<223> E . coli essential gene lolA

<400> 27 atg aaa aaa att gcc atc acc tgt gca tta ctc tca agc tta gta gca 48 Met Lys Lys Ile Ala Ile Thr Cys Ala Leu Leu Ser Ser Leu Val Ala 1 5 10 15 agc agc gtt tgg gct gat gcc gca agc gat ctg aaa agc cgc ctg gat 96 Ser Ser Val Trp Ala Asp Ala Ala Ser Asp Leu Lys Ser Arg Leu Asp 20 25 30 aaa gtc agc agc ttc cac gcc agc ttc aca caa aaa gtg act gac ggt 144 Lys Val Ser Ser Phe His Ala Ser Phe Thr Gin Lys Val Thr Asp Gly 35 40 45 agc ggc gcg gcg gtg cag gaa ggt cag ggc gat ctg tgg gtg aaa cgt 192 Ser Gly Ala Ala Val Gin Glu Gly Gin Gly Asp Leu Trp Val Lys Arg 50 55 60 cca aac tta ttc aac tgg cat atg aca caa cct gat gaa agc att ctg 240 Pro Asn Leu Phe Asn Trp His Met Thr Gin Pro Asp Glu Ser Ile Leu 65 70 75 80 gtt tct gac ggt aaa aca ctg tgg ttc tat aac ccg ttc gtt gag caa 288 Val Ser Asp Gly Lys Thr Leu Trp Phe Tyr Asn Pro Phe Val Glu Gin

85 90 95 gct acg gca acc tgg ctg aaa gat gcc acc ggt aat acg ccg ttt atg 336

Ala Thr Ala Thr Trp Leu Lys Asp Ala Thr Gly Asn Thr Pro Phe Met

100 105 110 ctg att gcc cgc aac cag tcc agc gac tgg cag cag tac aat atc aaa 384

Leu Ile Ala Arg Asn Gin Ser Ser Asp Trp Gin Gin Tyr Asn Ile Lys

115 120 125 cag aat ggc gat gac ttt gtc ctg acg ccg aaa gcc agc aat ggc aat 432

Gin Asn Gly Asp Asp Phe Val Leu Thr Pro Lys Ala Ser Asn Gly Asn

130 135 140 ctg aag cag ttc acc att aac gtg gga cgt gat ggc aca atc cat cag 480

Leu Lys Gin Phe Thr Ile Asn Val Gly Arg Asp Gly Thr Ile His Gin

145 150 155 160 ttt agc gcg gtg gag cag gac gat cag cgc agc agt tat caa ctg aaa 528

Phe Ser Ala Val Glu Gin Asp Asp Gin Arg Ser Ser Tyr Gin Leu Lys

165 170 175 tcc cag caa aat ggg gct gtg gat gca gcg aaa ttt acc ttc acc ccg 576

Ser Gin Gin Asn Gly Ala Val Asp Ala Ala Lys Phe Thr Phe Thr Pro

180 185 190 ccg caa ggc gtc acg gta gat gat caa cgt aag tag 612

Pro Gin Gly Val Thr Val Asp Asp Gin Arg Lys

195 200

<210> 28

<211> 203

<212> PRT

<213> E . coli

<400> 28

Met Lys Lys Ile Ala Ile Thr Cys Ala Leu Leu Ser Ser Leu Val Ala

1 5 10 15

Ser Ser Val Trp Ala Asp Ala Ala Ser Asp Leu Lys Ser Arg Leu Asp 20 25 30

Lys Val Ser Ser Phe His Ala Ser Phe Thr Gin Lys Val Thr Asp Gly 35 40 45 Ser Gly Ala Ala Val Gin Glu Gly Gin Gly Asp Leu Trp Val Lys Arg 50 55 60

Pro Asn Leu Phe Asn Trp His Met Thr Gin Pro Asp Glu Ser Ile Leu 65 70 75 80 Val Ser Asp Gly Lys Thr Leu Trp Phe Tyr Asn Pro Phe Val Glu Gin 85 90 95

Ala Thr Ala Thr Trp Leu Lys Asp Ala Thr Gly Asn Thr Pro Phe Met 100 105 110

Leu Ile Ala Arg Asn Gin Ser Ser Asp Trp Gin Gin Tyr Asn Ile Lys 115 120 125

Gin Asn Gly Asp Asp Phe Val Leu Thr Pro Lys Ala Ser Asn Gly Asn 130 135 140

Leu Lys Gin Phe Thr Ile Asn Val Gly Arg Asp Gly Thr Ile His Gin

145 150 155 160

Phe Ser Ala Val Glu Gin Asp Asp Gin Arg Ser Ser Tyr Gin Leu Lys 165 170 175

Ser Gin Gin Asn Gly Ala Val Asp Ala Ala Lys Phe Thr Phe Thr Pro 180 185 190

Pro Gin Gly Val Thr Val Asp Asp Gin Arg Lys 195 200

<210> 29

<211> 1083

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1083)

<223> E . coli essential gene mraY

<400> 29 atg tta gtt tgg ctg gcc gaa cat ttg gtc aaa tat tat tcc ggc ttt 48 Met Leu Val Trp Leu Ala Glu His Leu Val Lys Tyr Tyr Ser Gly Phe 1 5 10 15 aac gtc ttt tcc tat ctg acg ttt cgc gcc atc gtc agc ctg ctg acc 96 Asn Val Phe Ser Tyr Leu Thr Phe Arg Ala Ile Val Ser Leu Leu Thr 20 25 30 gcg ctg ttc atc tca ttg tgg atg ggc ccg cgt atg att gct cat ttg 144

Ala Leu Phe Ile Ser Leu Trp Met Gly Pro Arg Met Ile Ala His Leu

35 40 45 caa aaa ctt tcc ttt ggt cag gtg gtg cgt aac gac ggt cct gaa tca 192

Gin Lys Leu Ser Phe Gly Gin Val Val Arg Asn Asp Gly Pro Glu Ser

50 55 60 cac ttc agc aag cgc ggt acg ccg acc atg ggc ggg att atg atc ctg 240

His Phe Ser Lys Arg Gly Thr Pro Thr Met Gly Gly Ile Met Ile Leu

65 70 75 80 acg gcg att gtg atc tcc gta ctg ctg tgg gct tac ccg tec aat ccg 288

Thr Ala Ile Val Ile Ser Val Leu Leu Trp Ala Tyr Pro Ser Asn Pro

85 90 95 tac gtc tgg tgc gtg ttg gtg gtg ctg gta ggt tac ggt gtt att ggc 336

Tyr Val Trp Cys Val Leu Val Val Leu Val Gly Tyr Gly Val Ile Gly

100 105 110 ttt gtt gat gat tat cgc aaa gtg gtg cgt aaa gac acc aaa ggg ttg 384

Phe Val Asp Asp Tyr Arg Lys Val Val Arg Lys Asp Thr Lys Gly Leu

115 120 125 atc gct cgt tgg aag tat ttc tgg atg tcg gtc att gcg ctg ggt gtc 432 Ile Ala Arg Trp Lys Tyr Phe Trp Met Ser Val Ile Ala Leu Gly Val

130 135 140 gcc ttc gcc ctg tac ctt gcc ggc aaa gac acg ccc gca acg cag ctg 480

Ala Phe Ala Leu Tyr Leu Ala Gly Lys Asp Thr Pro Ala Thr Gin Leu

145 150 155 160 gtg gtc cca ttc ttt aaa gat gtg atg ccg cag ctg ggg ctg ttc tac 528 Val Val Pro Phe Phe Lys Asp Val Met Pro Gin Leu Gly Leu Phe Tyr

165 170 175 att ctg ctg gct tac ttc gtc att gtg ggt act ggc aac gcg gta aac 576 Ile Leu Leu Ala Tyr Phe Val Ile Val Gly Thr Gly Asn Ala Val Asn

180 185 190 ctg acc gat ggt ctc gac ggc ctg gca att atg ccg acc gta ttt gtc 624

Leu Thr Asp Gly Leu Asp Gly Leu Ala Ile Met Pro Thr Val Phe Val

195 200 205 gcc ggt ggt ttt gcg ctg gtg gcg tgg gcg acc ggc aat atg aac ttt 672

Ala Gly Gly Phe Ala Leu Val Ala Trp Ala Thr Gly Asn Met Asn Phe

210 215 220 gcc agc tac ttg cat ata ccg tat ctg cga cac gcc ggg gaa ctg gtt 720

Ala Ser Tyr Leu His Ile Pro Tyr Leu Arg His Ala Gly Glu Leu Val

225 230 235 240 att gtc tgt acc gcg ata gtc ggg gca gga ctg ggc ttc ctg tgg ttt 768 Ile Val Cys Thr Ala Ile Val Gly Ala Gly Leu Gly Phe Leu Trp Phe

245 250 255 aac acc tat ccg gcg cag gtc ttt atg ggc gat gta ggt tcg ctg gcg 816

Asn Thr Tyr Pro Ala Gin Val Phe Met Gly Asp Val Gly Ser Leu Ala

260 265 270 tta ggt ggt gcg tta ggc att atc gcc gta ctg cta cgt cag gaa ttc 864

Leu Gly Gly Ala Leu Gly Ile Ile Ala Val Leu Leu Arg Gin Glu Phe

275 280 285 ctg ctg gtg att atg ggg ggc gtg ttc gtg gta gaa acg ctt tct gtc 912 Leu Leu Val Ile Met Gly Gly Val Phe Val Val Glu Thr Leu Ser Val 290 295 300 atc ctg cag gtc ggc tcc ttt aaa ctg cgc gga caa cgt att ttc cgc 960 Ile Leu Gin Val Gly Ser Phe Lys Leu Arg Gly Gin Arg Ile Phe Arg 305 310 315 320 atg gca ccg att cat cac cac tat gaa ctg aaa ggc tgg ccg gaa ccg 1008 Met Ala Pro Ile His His His Tyr Glu Leu Lys Gly Trp Pro Glu Pro 325 330 335 cgc gtc att gtg cgt ttc tgg att att tcg ctg atg ctg gtt ctg att 1056 Arg Val Ile Val Arg Phe Trp Ile Ile Ser Leu Met Leu Val Leu Ile 340 345 350 ggt ctg gca acg ctg aag gta cgt taa 1083 Gly Leu Ala Thr Leu Lys Val Arg 355 360

<210> 30

<211> 360

<212> PRT

<213> E . coli

<400> 30

Met Leu Val Trp Leu Ala Glu His Leu Val Lys Tyr Tyr Ser Gly Phe

1 5 10 15

Asn Val Phe Ser Tyr Leu Thr Phe Arg Ala Ile Val Ser Leu Leu Thr 20 25 30

Ala Leu Phe Ile Ser Leu Trp Met Gly Pro Arg Met Ile Ala His Leu 35 40 45

Gin Lys Leu Ser Phe Gly Gin Val Val Arg Asn Asp Gly Pro Glu Ser 50 55 60

His Phe Ser Lys Arg Gly Thr Pro Thr Met Gly Gly Ile Met Ile Leu 65 70 75 80

Thr Ala Ile Val Ile Ser Val Leu Leu Trp Ala Tyr Pro Ser Asn Pro 85 90 95

Tyr Val Trp Cys Val Leu Val Val Leu Val Gly Tyr Gly Val Ile Gly 100 105 110

Phe Val Asp Asp Tyr Arg Lys Val Val Arg Lys Asp Thr Lys Gly Leu 115 120 125 Ile Ala Arg Trp Lys Tyr Phe Trp Met Ser Val Ile Ala Leu Gly Val

130 135 140

Ala Phe Ala Leu Tyr Leu Ala Gly Lys Asp Thr Pro Ala Thr Gin Leu 145 150 155 160 Val Val Pro Phe Phe Lys Asp Val Met Pro Gin Leu Gly Leu Phe Tyr 165 170 175 Ile Leu Leu Ala Tyr Phe Val Ile Val Gly Thr Gly Asn Ala Val Asn

180 185 190

Leu Thr Asp Gly Leu Asp Gly Leu Ala Ile Met Pro Thr Val Phe Val 195 200 205

Ala Gly Gly Phe Ala Leu Val Ala Trp Ala Thr Gly Asn Met Asn Phe

210 215 220

Ala Ser Tyr Leu His Ile Pro Tyr Leu Arg His Ala Gly Glu Leu Val 225 230 235 240 Ile Val Cys Thr Ala Ile Val Gly Ala Gly Leu Gly Phe Leu Trp Phe

245 250 255

Asn Thr Tyr Pro Ala Gin Val Phe Met Gly Asp Val Gly Ser Leu Ala

260 265 270

Leu Gly Gly Ala Leu Gly Ile Ile Ala Val Leu Leu Arg Gin Glu Phe 275 280 285

Leu Leu Val Ile Met Gly Gly Val Phe Val Val Glu Thr Leu Ser Val

290 295 300 Ile Leu Gin Val Gly Ser Phe Lys Leu Arg Gly Gin Arg Ile Phe Arg 305 310 315 320

Met Ala Pro Ile His His His Tyr Glu Leu Lys Gly Trp Pro Glu Pro 325 330 335

Arg Val Ile Val Arg Phe Trp Ile Ile Ser Leu Met Leu Val Leu Ile

340 345 350

Gly Leu Ala Thr Leu Lys Val Arg

355 360

<210> 31

<211> 489

<212> DNA

<213> E . coli

<220>

<221> CDS <222> (1) . . (489)

<223> E . coli essential gene mreD

<400> 31 gtg gcg agc tat cgt agc cag gga cgc tgg gta atc tgg ctc tct ttc 48 Val Ala Ser Tyr Arg Ser Gin Gly Arg Trp Val Ile Trp Leu Ser Phe 1 5 10 15 ctc att gcg ctg ttg ctg caa atc atg ccc tgg ccg gat aac ctg att 96

Leu Ile Ala Leu Leu Leu Gin Ile Met Pro Trp Pro Asp Asn Leu Ile

20 25 30 gtt ttc egg cca aac tgg gtg tta ctc atc ttg ttg tat tgg atc ctg 144 Val Phe Arg Pro Asn Trp Val Leu Leu Ile Leu Leu Tyr Trp Ile Leu

35 40 45 gcc ttg cct cat cgc gta aat gtg ggc aca ggt ttt gtg atg ggt gcc 192

Ala Leu Pro His Arg Val Asn Val Gly Thr Gly Phe Val Met Gly Ala

50 55 60 ata ctg gat ctg atc agc ggc tcg acg ctt ggc gta cgc gta ttg gcg 240 Ile Leu Asp Leu Ile Ser Gly Ser Thr Leu Gly Val Arg Val Leu Ala

65 70 75 80 atg agc atc att gct tac ctg gtg gcg ctg aaa tac cag ctt ttc cgc 288

Met Ser Ile Ile Ala Tyr Leu Val Ala Leu Lys Tyr Gin Leu Phe Arg

85 90 95 aac ctc gca tta tgg cag cag gcg ctg gtc gtc atg ttg ctt tcg ctg 336

Asn Leu Ala Leu Trp Gin Gin Ala Leu Val Val Met Leu Leu Ser Leu

100 105 110 gtg gtg gat att att gtt ttc tgg gca gag ttt tta gtg att aac gtc 384 Val Val Asp Ile Ile Val Phe Trp Ala Glu Phe Leu Val Ile Asn Val

115 120 125 tct ttc aga ccg gaa gtg ttc tgg agt agt gta gtc aat ggg gtg ctc 432

Ser Phe Arg Pro Glu Val Phe Trp Ser Ser Val Val Asn Gly Val Leu

130 135 140 tgg ccg tgg att ttc ttg ctg atg cgc aaa gtc cgt cag cag ttt gca 480

Trp Pro Trp Ile Phe Leu Leu Met Arg Lys Val Arg Gin Gin Phe Ala

145 150 155 160 gtg caa taa 489 Val Gin

<210> 32

<211> 162

<212> PRT

<213> E . coli

<400> 32 Val Ala Ser Tyr Arg Ser Gin Gly Arg Trp Val Ile Trp Leu Ser Phe

1 5 10 15

Leu Ile Ala Leu Leu Leu Gin Ile Met Pro Trp Pro Asp Asn Leu Ile 20 25 30 Val Phe Arg Pro Asn Trp Val Leu Leu Ile Leu Leu Tyr Trp Ile Leu 35 40 45

Ala Leu Pro His Arg Val Asn Val Gly Thr Gly Phe Val Met Gly Ala

50 55 60 Ile Leu Asp Leu Ile Ser Gly Ser Thr Leu Gly Val Arg Val Leu Ala 65 70 75 80

Met Ser Ile Ile Ala Tyr Leu Val Ala Leu Lys Tyr Gin Leu Phe Arg 85 90 95

Asn Leu Ala Leu Trp Gin Gin Ala Leu Val Val Met Leu Leu Ser Leu 100 105 110 Val Val Asp Ile Ile Val Phe Trp Ala Glu Phe Leu Val Ile Asn Val 115 120 125

Ser Phe Arg Pro Glu Val Phe Trp Ser Ser Val Val Asn Gly Val Leu

130 135 140

Trp Pro Trp Ile Phe Leu Leu Met Arg Lys Val Arg Gin Gin Phe Ala 145 150 155 160 Val Gin

<210> 33

<211> 1260

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1260)

<223> E . coli essential gene murA

<400> 33 atg gat aaa ttt cgt gtt cag ggg cca acg aag ctc cag ggc gaa gtc 48

Met Asp Lys Phe Arg Val Gin Gly Pro Thr Lys Leu Gin Gly Glu Val

1 5 10 15 aca att tcc ggc gct aaa aat gct gct ctg cct atc ctt ttt gcc gct 96

Thr Ile Ser Gly Ala Lys Asn Ala Ala Leu Pro Ile Leu Phe Ala Ala

20 25 30 cta ctg gcg gaa gaa ccg gta gag atc cag aac gtc ccg aaa ctg aaa 144

Leu Leu Ala Glu Glu Pro Val Glu Ile Gin Asn Val Pro Lys Leu Lys

35 40 45 gac gtc gat aca tca atg aag ctg cta agc cag ctg ggt gcg aaa gta 192

Asp Val Asp Thr Ser Met Lys Leu Leu Ser Gin Leu Gly Ala Lys Val 50 55 60 gaa cgt aat ggt tct gtg cat att gat gcc cgc gac gtt aat gta ttc 240

Glu Arg Asn Gly Ser Val His Ile Asp Ala Arg Asp Val Asn Val Phe

65 70 75 80 tgc gca cct tac gat ctg gtt aaa acc atg cgt gct tct atc tgg gca 288

Cys Ala Pro Tyr Asp Leu Val Lys Thr Met Arg Ala Ser Ile Trp Ala

85 90 95 ctg ggg ccg ctg gta gcg cgc ttt ggt cag ggg caa gtt tca cta cct 336

Leu Gly Pro Leu Val Ala Arg Phe Gly Gin Gly Gin Val Ser Leu Pro

100 105 110 ggc ggt tgt acg atc ggt gcg cgt ccg gtt gat cta cac att tct ggc 384

Gly Gly Cys Thr Ile Gly Ala Arg Pro Val Asp Leu His Ile Ser Gly

115 120 125 ctc gaa caa tta ggc gcg acc atc aaa ctg gaa gaa ggt tac gtt aaa 432

Leu Glu Gin Leu Gly Ala Thr Ile Lys Leu Glu Glu Gly Tyr Val Lys

130 135 140 gct tec gtc gat ggt cgt ctg aaa ggc gca cat atc gtg atg gat aaa 480

Ala Ser Val Asp Gly Arg Leu Lys Gly Ala His Ile Val Met Asp Lys

145 150 155 160 gtc agc gtt ggc gca acg gtg acc atc atg tgt gct gca acc ctg gcg 528 Val Ser Val Gly Ala Thr Val Thr Ile Met Cys Ala Ala Thr Leu Ala

165 170 175 gaa ggc acc acg att att gaa aac gca gcg cgt gaa ccg gaa atc gtc 576

Glu Gly Thr Thr Ile Ile Glu Asn Ala Ala Arg Glu Pro Glu Ile Val

180 185 190 gat acc gcg aac ttc ctg att acg ctg ggt gcg aaa att agc ggt cag 624

Asp Thr Ala Asn Phe Leu Ile Thr Leu Gly Ala Lys Ile Ser Gly Gin

195 200 205 ggc acc gat cgt atc gtc atc gaa ggt gtg gaa cgt tta ggc ggc ggt 672

Gly Thr Asp Arg Ile Val Ile Glu Gly Val Glu Arg Leu Gly Gly Gly

210 215 220 gtc tat cgc gtg ctg ccg gat cgt atc gaa acc ggt aca ttc ctg gtg 720 Val Tyr Arg Val Leu Pro Asp Arg Ile Glu Thr Gly Thr Phe Leu Val

225 230 235 240 gcg gcg gcg att tct cgc ggc aaa att atc tgc cgt aac gcg cag cca 768

Ala Ala Ala Ile Ser Arg Gly Lys Ile Ile Cys Arg Asn Ala Gin Pro

245 250 255 gat act ctc gac gcc gtg ctg gcg aaa ctg cgt gac gct gga gcg gac 816

Asp Thr Leu Asp Ala Val Leu Ala Lys Leu Arg Asp Ala Gly Ala Asp

260 265 270 atc gaa gtc ggc gaa gac tgg att agc ctg gat atg cat ggc aaa cgt 864 Ile Glu Val Gly Glu Asp Trp Ile Ser Leu Asp Met His Gly Lys Arg

275 280 285 ccg aag gct gtt aac gta cgt acc gcg ccg cat ccg gca ttc ccg acc 912

Pro Lys Ala Val Asn Val Arg Thr Ala Pro His Pro Ala Phe Pro Thr

290 295 300 gat atg cag gcc cag ttc acg ctg ttg aac ctg gtg gca gaa ggg act 960 Asp Met Gin Ala Gin Phe Thr Leu Leu Asn Leu Val Ala Glu Gly Thr 305 310 315 320 gga ttc atc acc gaa acg gtc ttt gaa aac cgc ttt atg cat gtg cca 1008

Gly Phe Ile Thr Glu Thr Val Phe Glu Asn Arg Phe Met His Val Pro

325 330 335 gag ctg agc cgt atg ggc gcg cac gcc gaa atc gaa agc aat acc gtt 1056

Glu Leu Ser Arg Met Gly Ala His Ala Glu Ile Glu Ser Asn Thr Val

340 345 350 att tgt cac ggt gtt gaa aaa ctt tct ggc gca cag gtt atg gca acc 1104 Ile Cys His Gly Val Glu Lys Leu Ser Gly Ala Gin Val Met Ala Thr

355 360 365 gat ctg cgt gca tca gca agc ctg gtg ctg gct ggc tgt att gcg gaa 1152

Asp Leu Arg Ala Ser Ala Ser Leu Val Leu Ala Gly Cys Ile Ala Glu

370 375 380 ggg acg acg gtg gtt gat cgt att tat cac atc gat cgt ggc tac gaa 1200

Gly Thr Thr Val Val Asp Arg Ile Tyr His Ile Asp Arg Gly Tyr Glu

385 390 395 400 cgc att gaa gac aaa ctg cgc gct tta ggt gca aat att gag cgt gtg 1248

Arg Ile Glu Asp Lys Leu Arg Ala Leu Gly Ala Asn Ile Glu Arg Val

405 410 415 aaa ggc gag taa 1260

Lys Gly Glu

<210> 34

<211> 419

<212> PRT

<213> E . coll

<400> 34

Met Asp Lys Phe Arg Val Gin Gly Pro Thr Lys Leu Gin Gly Glu Val

1 5 10 15

Thr Ile Ser Gly Ala Lys Asn Ala Ala Leu Pro Ile Leu Phe Ala Ala 20 25 30

Leu Leu Ala Glu Glu Pro Val Glu Ile Gin Asn Val Pro Lys Leu Lys 35 40 45

Asp Val Asp Thr Ser Met Lys Leu Leu Ser Gin Leu Gly Ala Lys Val 50 55 60

Glu Arg Asn Gly Ser Val His Ile Asp Ala Arg Asp Val Asn Val Phe 65 70 75 80

Cys Ala Pro Tyr Asp Leu Val Lys Thr Met Arg Ala Ser Ile Trp Ala 85 90 95 Leu Gly Pro Leu Val Ala Arg Phe Gly Gin Gly Gin Val Ser Leu Pro 100 105 110

Gly Gly Cys Thr Ile Gly Ala Arg Pro Val Asp Leu His Ile Ser Gly 115 120 125

Leu Glu Gin Leu Gly Ala Thr Ile Lys Leu Glu Glu Gly Tyr Val Lys 130 135 140

Ala Ser Val Asp Gly Arg Leu Lys Gly Ala His Ile Val Met Asp Lys 145 150 155 160 Val Ser Val Gly Ala Thr Val Thr Ile Met Cys Ala Ala Thr Leu Ala 165 170 175

Glu Gly Thr Thr Ile Ile Glu Asn Ala Ala Arg Glu Pro Glu Ile Val 180 185 190

Asp Thr Ala Asn Phe Leu Ile Thr Leu Gly Ala Lys Ile Ser Gly Gin 195 200 205

Gly Thr Asp Arg Ile Val Ile Glu Gly Val Glu Arg Leu Gly Gly Gly 210 215 220 Val Tyr Arg Val Leu Pro Asp Arg Ile Glu Thr Gly Thr Phe Leu Val 225 230 235 240

Ala Ala Ala Ile Ser Arg Gly Lys Ile Ile Cys Arg Asn Ala Gin Pro

245 250 255

Asp Thr Leu Asp Ala Val Leu Ala Lys Leu Arg Asp Ala Gly Ala Asp 260 265 270 Ile Glu Val Gly Glu Asp Trp Ile Ser Leu Asp Met His Gly Lys Arg 275 280 285

Pro Lys Ala Val Asn Val Arg Thr Ala Pro His Pro Ala Phe Pro Thr 290 295 300

Asp Met Gin Ala Gin Phe Thr Leu Leu Asn Leu Val Ala Glu Gly Thr 305 310 315 320

Gly Phe Ile Thr Glu Thr Val Phe Glu Asn Arg Phe Met His Val Pro 325 330 335

Glu Leu Ser Arg Met Gly Ala His Ala Glu Ile Glu Ser Asn Thr Val 340 345 350 Ile Cys His Gly Val Glu Lys Leu Ser Gly Ala Gin Val Met Ala Thr 355 360 365

Asp Leu Arg Ala Ser Ala Ser Leu Val Leu Ala Gly Cys Ile Ala Glu

370 375 380

Gly Thr Thr Val Val Asp Arg Ile Tyr His Ile Asp Arg Gly Tyr Glu 385 390 395 400

Arg Ile Glu Asp Lys Leu Arg Ala Leu Gly Ala Asn Ile Glu Arg Val

405 410 415

Lys Gly Glu

<210> 35

<211> 1029

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1029)

<223> E . coli essential gene murB

<400> 35 atg aac cac tcc tta aaa ccc tgg aac aca ttt ggc att gat cat aat 48

Met Asn His Ser Leu Lys Pro Trp Asn Thr Phe Gly Ile Asp His Asn

1 5 10 15 gct cag cac att gta tgt gcc gaa gac gaa caa caa tta ctc aat gcc 96

Ala Gin His Ile Val Cys Ala Glu Asp Glu Gin Gin Leu Leu Asn Ala

20 25 30 tgg cag cat gca acc gca gaa gga caa ccc gtt ctt att ctg ggt gaa 144 Trp Gin His Ala Thr Ala Glu Gly Gin Pro Val Leu Ile Leu Gly Glu 35 40 45 gga agt aat gta ctt ttt ctg gaa gac tat cgc ggc acg gtg atc atc 192 Gly Ser Asn Val Leu Phe Leu Glu Asp Tyr Arg Gly Thr Val Ile Ile 50 55 60 aac egg atc aaa ggt atc gaa att cat gat gaa cct gat gcg tgg tat 240 Asn Arg Ile Lys Gly Ile Glu Ile His Asp Glu Pro Asp Ala Trp Tyr 65 70 75 80 tta cat gta gga gcc gga gaa aac tgg cat cgc ctg gta aaa tac act 288 Leu His Val Gly Ala Gly Glu Asn Trp His Arg Leu Val Lys Tyr Thr 85 90 95 ttg cag gaa ggt atg cct ggt ctg gaa aat ctg gca tta att cct ggt 336

Leu Gin Glu Gly Met Pro Gly Leu Glu Asn Leu Ala Leu Ile Pro Gly 100 105 110 tgt gtc ggc tca tca cct atc cag aat att ggt gct tat ggc gta gaa 384 Cys Val Gly Ser Ser Pro Ile Gin Asn Ile Gly Ala Tyr Gly Val Glu 115 120 125 tta cag cga gtt tgc gct tat gtt gat tgt gtt gaa ctg gcg aca ggc 432

Leu Gin Arg Val Cys Ala Tyr Val Asp Cys Val Glu Leu Ala Thr Gly

130 135 140 aag caa gtg cgc tta act gcc aaa gag tgc cgt ttt ggc tat cgc gac 480

Lys Gin Val Arg Leu Thr Ala Lys Glu Cys Arg Phe Gly Tyr Arg Asp

145 150 155 160 agt att ttt aaa cat gaa tac cag gat cgc ttc gcc att gta gcc gta 528

Ser Ile Phe Lys His Glu Tyr Gin Asp Arg Phe Ala Ile Val Ala Val

165 170 175 ggt ctg cgt ctg cca aaa gag tgg caa cct gta cta acg tat ggt gac 576

Gly Leu Arg Leu Pro Lys Glu Trp Gin Pro Val Leu Thr Tyr Gly Asp

180 185 190 tta act cgt ctg gat cct aca aca gta acg cca cag caa gta ttt aat 624

Leu Thr Arg Leu Asp Pro Thr Thr Val Thr Pro Gin Gin Val Phe Asn

195 200 205 gcg gtg tgt cat atg cgc acc acc aaa ctc cct gat cca aaa gtg aat 672

Ala Val Cys His Met Arg Thr Thr Lys Leu Pro Asp Pro Lys Val Asn

210 215 220 ggc aat gcc ggt agt ttc ttc aaa aac cct gtt gta tct gcc gaa acg 720

Gly Asn Ala Gly Ser Phe Phe Lys Asn Pro Val Val Ser Ala Glu Thr

225 230 235 240 gct gaa gca tta ctg tca caa ttt cca aca gca cca aat tac ccc cag 768

Ala Glu Ala Leu Leu Ser Gin Phe Pro Thr Ala Pro Asn Tyr Pro Gin

245 250 255 gcg gat ggt tca gta aaa ctg gca gca ggt tgg ctt att gat cag tgc 816

Ala Asp Gly Ser Val Lys Leu Ala Ala Gly Trp Leu Ile Asp Gin Cys

260 265 270 cag cta aaa ggg atg caa atg ggt ggg gct gcg gtg cac cgt caa cag 864

Gin Leu Lys Gly Met Gin Met Gly Gly Ala Ala Val His Arg Gin Gin

275 280 285 gcg tta gtt ctc att aat gaa gac aat gca aaa agc gaa gat gtg gtg 912

Ala Leu Val Leu Ile Asn Glu Asp Asn Ala Lys Ser Glu Asp Val Val

290 295 300 caa ctg gca cac cat gta aga caa aaa gtg ggt gaa aaa ttt aat gtc 960

Gin Leu Ala His His Val Arg Gin Lys Val Gly Glu Lys Phe Asn Val

305 310 315 320 tgg ctt gag cct gaa gtc cgc ttt att ggt gca tca ggt gaa gtg agc 1008

Trp Leu Glu Pro Glu Val Arg Phe Ile Gly Ala Ser Gly Glu Val Ser

325 330 335 gca gtg gag aca att tca tga 1029

Ala Val Glu Thr Ile Ser

340

<210> 36

<211> 342

<212> PRT

<213> E . coli <400> 36

Met Asn His Ser Leu Lys Pro Trp Asn Thr Phe Gly Ile Asp His Asn 1 5 10 15

Ala Gin His Ile Val Cys Ala Glu Asp Glu Gin Gin Leu Leu Asn Ala 20 25 30

Trp Gin His Ala Thr Ala Glu Gly Gin Pro Val Leu Ile Leu Gly Glu 35 40 45

Gly Ser Asn Val Leu Phe Leu Glu Asp Tyr Arg Gly Thr Val Ile Ile 50 55 60

Asn Arg Ile Lys Gly Ile Glu Ile His Asp Glu Pro Asp Ala Trp Tyr 65 70 75 80

Leu His Val Gly Ala Gly Glu Asn Trp His Arg Leu Val Lys Tyr Thr 85 90 95

Leu Gin Glu Gly Met Pro Gly Leu Glu Asn Leu Ala Leu Ile Pro Gly 100 105 110

Cys Val Gly Ser Ser Pro Ile Gin Asn Ile Gly Ala Tyr Gly Val Glu 115 120 125

Leu Gin Arg Val Cys Ala Tyr Val Asp Cys Val Glu Leu Ala Thr Gly 130 135 140

Lys Gin Val Arg Leu Thr Ala Lys Glu Cys Arg Phe Gly Tyr Arg Asp

145 150 155 160

Ser Ile Phe Lys His Glu Tyr Gin Asp Arg Phe Ala Ile Val Ala Val 165 170 175

Gly Leu Arg Leu Pro Lys Glu Trp Gin Pro Val Leu Thr Tyr Gly Asp 180 185 190

Leu Thr Arg Leu Asp Pro Thr Thr Val Thr Pro Gin Gin Val Phe Asn 195 200 205

Ala Val Cys His Met Arg Thr Thr Lys Leu Pro Asp Pro Lys Val Asn 210 215 220

Gly Asn Ala Gly Ser Phe Phe Lys Asn Pro Val Val Ser Ala Glu Thr

225 230 235 240 Ala Glu Ala Leu Leu Ser Gin Phe Pro Thr Ala Pro Asn Tyr Pro Gin

245 250 255

Ala Asp Gly Ser Val Lys Leu Ala Ala Gly Trp Leu Ile Asp Gin Cys 260 265 270

Gin Leu Lys Gly Met Gin Met Gly Gly Ala Ala Val His Arg Gin Gin 275 280 285

Ala Leu Val Leu Ile Asn Glu Asp Asn Ala Lys Ser Glu Asp Val Val 290 295 300

Gin Leu Ala His His Val Arg Gin Lys Val Gly Glu Lys Phe Asn Val 305 310 315 320

Trp Leu Glu Pro Glu Val Arg Phe Ile Gly Ala Ser Gly Glu Val Ser

325 330 335

Ala Val Glu Thr Ile Ser 340

<210> 37

<211> 1359

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (1359)

<223> E . coli essential gene murF

<400> 37 atg att agc gta acc ctt agc caa ctt acc gac att ctc aac ggt gaa 48 Met Ile Ser Val Thr Leu Ser Gin Leu Thr Asp Ile Leu Asn Gly Glu 1 5 10 15 ctg caa ggt gca gat atc acc ctt gat gct gta acc act gat acc cga 96 Leu Gin Gly Ala Asp Ile Thr Leu Asp Ala Val Thr Thr Asp Thr Arg 20 25 30 aaa ctg acg ccg ggc tgc ctg ttt gtt gcc ctg aaa ggc gaa cgt ttt 144 Lys Leu Thr Pro Gly Cys Leu Phe Val Ala Leu Lys Gly Glu Arg Phe 35 40 45 gat gcc cac gat ttt gcc gac cag gcg aaa gct ggc ggc gca ggc gca 192

Asp Ala His Asp Phe Ala Asp Gin Ala Lys Ala Gly Gly Ala Gly Ala

50 55 60 cta ctg gtt agc cgt ccg ctg gac atc gac ctg ccg cag tta atc gtc 240 Leu Leu Val Ser Arg Pro Leu Asp Ile Asp Leu Pro Gin Leu Ile Val 65 70 75 80 aag gat acg cgt ctg gcg ttt ggt gaa ctg gct gca tgg gtt cgc cag 288 Lys Asp Thr Arg Leu Ala Phe Gly Glu Leu Ala Ala Trp Val Arg Gin 85 90 95 caa gtt ccg gcg cgc gtg gtt gct ctg acg ggg tcc tcc ggc aaa acc 336 Gin Val Pro Ala Arg Val Val Ala Leu Thr Gly Ser Ser Gly Lys Thr 100 105 110 tcc gtt aaa gag atg acg gcg gcg att tta agc cag tgc ggc aac acg 384

Ser Val Lys Glu Met Thr Ala Ala Ile Leu Ser Gin Cys Gly Asn Thr

115 120 125 ctt tat acg gca ggc aat ctc aac aac gac atc ggt gta ccg atg acg 432

Leu Tyr Thr Ala Gly Asn Leu Asn Asn Asp Ile Gly Val Pro Met Thr

130 135 140 ctg ttg cgc tta acg ccg gaa tac gat tac gca gtt att gaa ctt ggc 480

Leu Leu Arg Leu Thr Pro Glu Tyr Asp Tyr Ala Val Ile Glu Leu Gly

145 150 155 160 gcg aac cat cag ggc gaa ata gcc tgg act gtg agt ctg act cgc ccg 528

Ala Asn His Gin Gly Glu Ile Ala Trp Thr Val Ser Leu Thr Arg Pro

165 170 175 gaa gct gcg ctg gtc aac aac ctg gca gcg gcg cat ctg gaa ggt ttt 576

Glu Ala Ala Leu Val Asn Asn Leu Ala Ala Ala His Leu Glu Gly Phe

180 185 190 ggc tcg ctt gcg ggt gtc gcg aaa gcg aaa ggt gaa atc ttt agc ggc 624 Gly Ser Leu Ala Gly Val Ala Lys Ala Lys Gly Glu Ile Phe Ser Gly 195 200 205 ctg ccg gaa aac ggt atc gcc att atg aac gcc gac aac aac gac tgg 672

Leu Pro Glu Asn Gly Ile Ala Ile Met Asn Ala Asp Asn Asn Asp Trp

210 215 220 ctg aac tgg cag agc gta att ggc tca cgc aaa gtg tgg cgt ttc tca 720

Leu Asn Trp Gin Ser Val Ile Gly Ser Arg Lys Val Trp Arg Phe Ser

225 230 235 240 ccc aat gcc gcc aac agc gat ttc acc gcc acc aat atc cat gtg acc 768

Pro Asn Ala Ala Asn Ser Asp Phe Thr Ala Thr Asn Ile His Val Thr

245 250 255 tcg cac ggt acg gaa ttt acc cta caa acc cca acc ggt agc gtc gat 816

Ser His Gly Thr Glu Phe Thr Leu Gin Thr Pro Thr Gly Ser Val Asp

260 265 270 gtt ctg ctg ccg ttg ccg ggg cgt cac aat att gcg aat gcg ctg gca 864 Val Leu Leu Pro Leu Pro Gly Arg His Asn Ile Ala Asn Ala Leu Ala

275 280 285 gcc gct gcg ctc tcc atg tcc gtg ggc gca acg ctt gat gct atc aaa 912

Ala Ala Ala Leu Ser Met Ser Val Gly Ala Thr Leu Asp Ala Ile Lys

290 295 300 gcg ggg ctg gca aat ctg aaa gct gtt cca ggc cgt ctg ttc ccc atc 960 Ala Gly Leu Ala Asn Leu Lys Ala Val Pro Gly Arg Leu Phe Pro Ile 305 310 315 320 caa ctg gca gaa aac cag ttg ctg ctc gac gac tcc tac aac gcc aat 1008 Gin Leu Ala Glu Asn Gin Leu Leu Leu Asp Asp Ser Tyr Asn Ala Asn 325 330 335 gtc ggt tca atg act gca gca gtc cag gta ctg gct gaa atg ccg ggc 1056 Val Gly Ser Met Thr Ala Ala Val Gin Val Leu Ala Glu Met Pro Gly 340 345 350 tac cgc gtg ctg gtg gtg ggc gat atg gcg gaa ctg ggc gct gaa agc 1104

Tyr Arg Val Leu Val Val Gly Asp Met Ala Glu Leu Gly Ala Glu Ser

355 360 365 gaa gcc tgc cat gta cag gtg ggc gag gcg gca aaa gct gct ggt att 1152

Glu Ala Cys His Val Gin Val Gly Glu Ala Ala Lys Ala Ala Gly Ile

370 375 380 gac cgc gtg tta agc gtg ggt aaa caa agc cat gct atc agc acc gcc 1200

Asp Arg Val Leu Ser Val Gly Lys Gin Ser His Ala Ile Ser Thr Ala

385 390 395 400 agc ggc gtt ggc gaa cat ttt gct gat aaa act gcg tta att acg cgt 1248

Ser Gly Val Gly Glu His Phe Ala Asp Lys Thr Ala Leu Ile Thr Arg

405 410 415 ctt aaa tca ctg att gct gag caa cag gta att acg att tta gtt aag 1296

Leu Lys Ser Leu Ile Ala Glu Gin Gin Val Ile Thr Ile Leu Val Lys

420 425 430 ggt tca cgt agt gcc gcc atg gaa gag gta gta cgc gct tta cag gag 1344

Gly Ser Arg Ser Ala Ala Met Glu Glu Val Val Arg Ala Leu Gin Glu

435 440 445 aat ggg aca tgt tag 1359

Asn Gly Thr Cys 450

<210> 38

<211> 452

<212> PRT

<213> E . coli

<400> 38

Met Ile Ser Val Thr Leu Ser Gin Leu Thr Asp Ile Leu Asn Gly Glu

1 5 10 15

Leu Gin Gly Ala Asp Ile Thr Leu Asp Ala Val Thr Thr Asp Thr Arg 20 25 30

Lys Leu Thr Pro Gly Cys Leu Phe Val Ala Leu Lys Gly Glu Arg Phe 35 40 45

Asp Ala His Asp Phe Ala Asp Gin Ala Lys Ala Gly Gly Ala Gly Ala 50 55 60

Leu Leu Val Ser Arg Pro Leu Asp Ile Asp Leu Pro Gin Leu Ile Val 65 70 75 80

Lys Asp Thr Arg Leu Ala Phe Gly Glu Leu Ala Ala Trp Val Arg Gin 85 90 95

Gin Val Pro Ala Arg Val Val Ala Leu Thr Gly Ser Ser Gly Lys Thr 100 105 110

Ser Val Lys Glu Met Thr Ala Ala Ile Leu Ser Gin Cys Gly Asn Thr 115 120 125

Leu Tyr Thr Ala Gly Asn Leu Asn Asn Asp Ile Gly Val Pro Met Thr 130 135 140

Leu Leu Arg Leu Thr Pro Glu Tyr Asp Tyr Ala Val Ile Glu Leu Gly

145 150 155 160

Ala Asn His Gin Gly Glu Ile Ala Trp Thr Val Ser Leu Thr Arg Pro 165 170 175

Glu Ala Ala Leu Val Asn Asn Leu Ala Ala Ala His Leu Glu Gly Phe 180 185 190

Gly Ser Leu Ala Gly Val Ala Lys Ala Lys Gly Glu Ile Phe Ser Gly 195 200 205

Leu Pro Glu Asn Gly Ile Ala Ile Met Asn Ala Asp Asn Asn Asp Trp 210 215 220

Leu Asn Trp Gin Ser Val Ile Gly Ser Arg Lys Val Trp Arg Phe Ser

225 230 235 240

Pro Asn Ala Ala Asn Ser Asp Phe Thr Ala Thr Asn Ile His Val Thr

245 250 255

Ser His Gly Thr Glu Phe Thr Leu Gin Thr Pro Thr Gly Ser Val Asp 260 265 270 Val Leu Leu Pro Leu Pro Gly Arg His Asn Ile Ala Asn Ala Leu Ala 275 280 285

Ala Ala Ala Leu Ser Met Ser Val Gly Ala Thr Leu Asp Ala Ile Lys 290 295 300

Ala Gly Leu Ala Asn Leu Lys Ala Val Pro Gly Arg Leu Phe Pro Ile 305 310 315 320

Gin Leu Ala Glu Asn Gin Leu Leu Leu Asp Asp Ser Tyr Asn Ala Asn 325 330 335 Val Gly Ser Met Thr Ala Ala Val Gin Val Leu Ala Glu Met Pro Gly 340 345 350 Tyr Arg Val Leu Val Val Gly Asp Met Ala Glu Leu Gly Ala Glu Ser 355 360 365

Glu Ala Cys His Val Gin Val Gly Glu Ala Ala Lys Ala Ala Gly Ile

370 375 380

Asp Arg Val Leu Ser Val Gly Lys Gin Ser His Ala Ile Ser Thr Ala 385 390 395 400

Ser Gly Val Gly Glu His Phe Ala Asp Lys Thr Ala Leu Ile Thr Arg

405 410 415

Leu Lys Ser Leu Ile Ala Glu Gin Gin Val Ile Thr Ile Leu Val Lys 420 425 430

Gly Ser Arg Ser Ala Ala Met Glu Glu Val Val Arg Ala Leu Gin Glu

435 440 445

Asn Gly Thr Cys

450

<210> 39

<211> 642

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . ( 642 )

<223> E . coli essential gene nadD

<400> 39 atg aaa tct tta cag gct ctg ttt ggc ggc acc ttt gat ccg gtg cac 48 Met Lys Ser Leu Gin Ala Leu Phe Gly Gly Thr Phe Asp Pro Val His 1 5 10 15 tat ggt cat cta aaa ccc gtg gaa acg ctg gcg aat ttg att ggt ctg 96

Tyr Gly His Leu Lys Pro Val Glu Thr Leu Ala Asn Leu Ile Gly Leu

20 25 30 acg egg gtc aca atc atc cct aat aat gtt cct ccg cat cgt ccc cag 144

Thr Arg Val Thr Ile Ile Pro Asn Asn Val Pro Pro His Arg Pro Gin

35 40 45 ccg gaa gcg aac agc gtg cag cgt aaa cac atg ctt gaa ctg gcg att 192

Pro Glu Ala Asn Ser Val Gin Arg Lys His Met Leu Glu Leu Ala Ile

50 55 60 gcc gac aag cca tta ttt act ctt gat gaa cgc gag cta aag cgc aat 240

Ala Asp Lys Pro Leu Phe Thr Leu Asp Glu Arg Glu Leu Lys Arg Asn

65 70 75 80 gcc ccc tct tac act gcg caa aca ctg aaa gag tgg egg cag gaa caa 288

Ala Pro Ser Tyr Thr Ala Gin Thr Leu Lys Glu Trp Arg Gin Glu Gin

85 90 95 gga ccg gac gtg ccg ctg gcg ttt att att ggt cag gat tca ttg ctg 336 Gly Pro Asp Val Pro Leu Ala Phe Ile Ile Gly Gin Asp Ser Leu Leu 100 105 110 acc ttt ccg acc tgg tac gaa tac gaa acg ata ctc gac aat gca cat 384

Thr Phe Pro Thr Trp Tyr Glu Tyr Glu Thr Ile Leu Asp Asn Ala His

115 120 125 ttg atc gtc tgt egg cgt cca ggt tac cca ctt gaa atg gcg caa ccg 432

Leu Ile Val Cys Arg Arg Pro Gly Tyr Pro Leu Glu Met Ala Gin Pro

130 135 140 caa tac cag caa tgg ctg gaa gat cat ttg aca cat aac ccg gaa gat 480

Gin Tyr Gin Gin Trp Leu Glu Asp His Leu Thr His Asn Pro Glu Asp

145 150 155 160 ctt cac ctt cag cct gcc ggt aaa att tat ctg gct gaa acg ccg tgg 528

Leu His Leu Gin Pro Ala Gly Lys Ile Tyr Leu Ala Glu Thr Pro Trp

165 170 175 ttt aac atc tcg gcg acc atc atc cgc gaa cgt ttg caa aac ggt gaa 576

Phe Asn Ile Ser Ala Thr Ile Ile Arg Glu Arg Leu Gin Asn Gly Glu

180 185 190 tcg tgt gag gat tta ttg ccg gaa ccg gta ttg act tac att aac caa 624

Ser Cys Glu Asp Leu Leu Pro Glu Pro Val Leu Thr Tyr Ile Asn Gin

195 200 205 caa ggc ttg tat cgc tga 642

Gin Gly Leu Tyr Arg

210

<210> 40

<211> 213

<212> PRT

<213> E . coll

<400> 40

Met Lys Ser Leu Gin Ala Leu Phe Gly Gly Thr Phe Asp Pro Val His

10 15

Tyr Gly His Leu Lys Pro Val Glu Thr Leu Ala Asn Leu Ile Gly Leu 20 25 30

Thr Arg Val Thr Ile Ile Pro Asn Asn Val Pro Pro His Arg Pro Gin 35 40 45

Pro Glu Ala Asn Ser Val Gin Arg Lys His Met Leu Glu Leu Ala Ile

50 55 60

Ala Asp Lys Pro Leu Phe Thr Leu Asp Glu Arg Glu Leu Lys Arg Asn 65 70 75 80

Ala Pro Ser Tyr Thr Ala Gin Thr Leu Lys Glu Trp Arg Gin Glu Gin 85 90 95 Gly Pro Asp Val Pro Leu Ala Phe Ile Ile Gly Gin Asp Ser Leu Leu 100 105 110

Thr Phe Pro Thr Trp Tyr Glu Tyr Glu Thr Ile Leu Asp Asn Ala His 115 120 125

Leu Ile Val Cys Arg Arg Pro Gly Tyr Pro Leu Glu Met Ala Gin Pro

130 135 140

Gin Tyr Gin Gin Trp Leu Glu Asp His Leu Thr His Asn Pro Glu Asp 145 150 155 160

Leu His Leu Gin Pro Ala Gly Lys Ile Tyr Leu Ala Glu Thr Pro Trp

165 170 175

Phe Asn Ile Ser Ala Thr Ile Ile Arg Glu Arg Leu Gin Asn Gly Glu 180 185 190

Ser Cys Glu Asp Leu Leu Pro Glu Pro Val Leu Thr Tyr Ile Asn Gin 195 200 205

Gin Gly Leu Tyr Arg

210

<210> 41

<211> 333

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (333)

<223> E . coli essential gene rplV

<400> 41 atg gaa act atc gct aaa cat cgc cat gct cgt tct tct gct cag aag 48

Met Glu Thr Ile Ala Lys His Arg His Ala Arg Ser Ser Ala Gin Lys

1 5 10 15 gtt cgc ctt gtt gct gac ctg att cgc ggt aag aaa gtg tcg cag gct 96 Val Arg Leu Val Ala Asp Leu Ile Arg Gly Lys Lys Val Ser Gin Ala

20 25 30 ctg gat att ttg acc tac acc aac aag aaa gcg gct gta ctg gtc aag 144

Leu Asp Ile Leu Thr Tyr Thr Asn Lys Lys Ala Ala Val Leu Val Lys

35 40 45 aaa gtt ctg gaa tct gcc att gct aac gct gaa cac aac gat ggc gct 192

Lys Val Leu Glu Ser Ala Ile Ala Asn Ala Glu His Asn Asp Gly Ala

50 55 60 gac att gac gat ctg aaa gtt acg aaa att ttc gta gac gaa ggc ccg 240 Asp Ile Asp Asp Leu Lys Val Thr Lys Ile Phe Val Asp Glu Gly Pro 65 70 75 80 agc atg aag cgc att atg ccg cgt gca aaa ggt cgt gca gat cgc atc 288

Ser Met Lys Arg Ile Met Pro Arg Ala Lys Gly Arg Ala Asp Arg Ile 85 90 95 ctg aag cgc acc agc cac atc act gtg gtt gtg tcc gat cgc tga 333

Leu Lys Arg Thr Ser His Ile Thr Val Val Val Ser Asp Arg 100 105 110

<210> 42

<211> 110

<212> PRT

<213> E . coli

<400> 42

Met Glu Thr Ile Ala Lys His Arg His Ala Arg Ser Ser Ala Gin Lys

1 5 10 15 Val Arg Leu Val Ala Asp Leu Ile Arg Gly Lys Lys Val Ser Gin Ala 20 25 30

Leu Asp Ile Leu Thr Tyr Thr Asn Lys Lys Ala Ala Val Leu Val Lys 35 40 45

Lys Val Leu Glu Ser Ala Ile Ala Asn Ala Glu His Asn Asp Gly Ala 50 55 60

Asp Ile Asp Asp Leu Lys Val Thr Lys Ile Phe Val Asp Glu Gly Pro 65 70 75 80

Ser Met Lys Arg Ile Met Pro Arg Ala Lys Gly Arg Ala Asp Arg Ile 85 90 95

Leu Lys Arg Thr Ser His Ile Thr Val Val Val Ser Asp Arg 100 105 110

<210> 43

<211> 471

<212> DNA

<213> E . coli

<220>

<221> CDS

<222> (1) . . (471)

<223> E . coli essential gene rpsG

<400> 43 atg cca cgt cgt cgc gtc att ggt cag cgt aaa att ctg ccg gat ccg 48 Met Pro Arg Arg Arg Val Ile Gly Gin Arg Lys Ile Leu Pro Asp Pro 1 5 10 15 aag ttc gga tca gaa ctg ctg gct aaa ttt gta aat atc ctg atg gta 96

Lys Phe Gly Ser Glu Leu Leu Ala Lys Phe Val Asn Ile Leu Met Val

20 25 30 gat ggt aaa aaa tct act gct gaa tct atc gta tac agc gcg ctg gag 144

Asp Gly Lys Lys Ser Thr Ala Glu Ser Ile Val Tyr Ser Ala Leu Glu

35 40 45 acc ctg gct cag cgc tct ggt aaa tct gaa ctg gaa gca ttc gaa gta 192

Thr Leu Ala Gin Arg Ser Gly Lys Ser Glu Leu Glu Ala Phe Glu Val

50 55 60 gct ctc gaa aac gtg cgc ccg act gta gaa gtt aag tct cgc cgc gtt 240

Ala Leu Glu Asn Val Arg Pro Thr Val Glu Val Lys Ser Arg Arg Val

65 70 75 80 ggt ggt tct act tat cag gta cca gtt gaa gtc cgt ccg gtt cgt cgt 288

Gly Gly Ser Thr Tyr Gin Val Pro Val Glu Val Arg Pro Val Arg Arg

85 90 95 aat gct ctg gca atg cgt tgg atc gtt gaa gct gct cgt aaa cgc ggt 336

Asn Ala Leu Ala Met Arg Trp Ile Val Glu Ala Ala Arg Lys Arg Gly

100 105 110 gat aaa tcc atg gct ctg cgc ctg gcg aac gaa ctt tct gat gct gca 384

Asp Lys Ser Met Ala Leu Arg Leu Ala Asn Glu Leu Ser Asp Ala Ala

115 120 125 gaa aac aaa ggt act gca gtt aag aaa cgt gaa gac gtt cac cgt atg 432

Glu Asn Lys Gly Thr Ala Val Lys Lys Arg Glu Asp Val His Arg Met

130 135 140 gcc gaa gcc aac aag gcg ttc gca cac tac cgt tgg taa 471

Ala Glu Ala Asn Lys Ala Phe Ala His Tyr Arg Trp 145 150 155

<210> 44

<211> 156

<212> PRT

<213> E . coli

<400> 44

Met Pro Arg Arg Arg Val Ile Gly Gin Arg Lys Ile Leu Pro Asp Pro

1 5 10 15

Lys Phe Gly Ser Glu Leu Leu Ala Lys Phe Val Asn Ile Leu Met Val 20 25 30

Asp Gly Lys Lys Ser Thr Ala Glu Ser Ile Val Tyr Ser Ala Leu Glu 35 40 45

Thr Leu Ala Gin Arg Ser Gly Lys Ser Glu Leu Glu Ala Phe Glu Val 50 55 60

Ala Leu Glu Asn Val Arg Pro Thr Val Glu Val Lys Ser Arg Arg Val 65 70 75 80 Gly Gly Ser Thr Tyr Gin Val Pro Val Glu Val Arg Pro Val Arg Arg 85 90 95

Asn Ala Leu Ala Met Arg Trp Ile Val Glu Ala Ala Arg Lys Arg Gly 100 105 110

Asp Lys Ser Met Ala Leu Arg Leu Ala Asn Glu Leu Ser Asp Ala Ala 115 120 125

Glu Asn Lys Gly Thr Ala Val Lys Lys Arg Glu Asp Val His Arg Met

130 135 140

Ala Glu Ala Asn Lys Ala Phe Ala His Tyr Arg Trp 145 150 155

<210> 45

<211> 2475

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . (2475)

<223> S . cerevisiae essential gene FOL1 YNL256W SGD1D : S000005200

<400> 45 atg tca aag cta ttt tct act gtc aat tct gca aga cat agt gta cca 48 Met Ser Lys Leu Phe Ser Thr Val Asn Ser Ala Arg His Ser Val Pro 1 5 10 15 cta ggc ggc atg aga gat tat gtg cac att aag aaa cta gag atg aat 96 Leu Gly Gly Met Arg Asp Tyr Val His Ile Lys Lys Leu Glu Met Asn 20 25 30 aca gtt ctt ggg cct gat tec tgg aat caa tta atg cct cag aaa tgt 144 Thr Val Leu Gly Pro Asp Ser Trp Asn Gin Leu Met Pro Gin Lys Cys 35 40 45 cta cta agc tta gat atg ggt aca gat ttt agt aaa tct gcg gct acg 192

Leu Leu Ser Leu Asp Met Gly Thr Asp Phe Ser Lys Ser Ala Ala Thr 50 55 60 gat gat ttg aaa tat tct cta aat tat gca gtt att tct cgt gat ttg 240 Asp Asp Leu Lys Tyr Ser Leu Asn Tyr Ala Val Ile Ser Arg Asp Leu 65 70 75 80 acg aat ttc gtc agc aaa aaa aag aat tgg ggt tct gtt tct aat ttg 288 Thr Asn Phe Val Ser Lys Lys Lys Asn Trp Gly Ser Val Ser Asn Leu 85 90 95 gct aaa tct gtg tct caa ttt gtt atg gac aaa tat tct ggt gtc gag 336

Ala Lys Ser Val Ser Gin Phe Val Met Asp Lys Tyr Ser Gly Val Glu 100 105 110 tgt ctg aat tta gaa gtg cag gcg gat aca acg cat att aga agt gac 384 Cys Leu Asn Leu Glu Val Gin Ala Asp Thr Thr His Ile Arg Ser Asp 115 120 125 cac ata tct tgt att att caa caa gaa aga ggg aat cca gaa tca cag 432

His Ile Ser Cys Ile Ile Gin Gin Glu Arg Gly Asn Pro Glu Ser Gin

130 135 140 gaa ttt gac gtt gtt agg ata tct gag tta aaa atg ttg act ttg att 480

Glu Phe Asp Val Val Arg Ile Ser Glu Leu Lys Met Leu Thr Leu Ile

145 150 155 160 ggt gtt ttc acc ttt gag aga ctt aag aaa cag tat gta act ttg gat 528

Gly Val Phe Thr Phe Glu Arg Leu Lys Lys Gin Tyr Val Thr Leu Asp

165 170 175 ata aag ttg cct tgg cca aag aaa gcc gaa ttg cca ccg cca gtg caa 576 Ile Lys Leu Pro Trp Pro Lys Lys Ala Glu Leu Pro Pro Pro Val Gin

180 185 190 agc ata att gat aac gtt gtc aag ttt gtg gag gaa tca aat ttc aag 624

Ser Ile Ile Asp Asn Val Val Lys Phe Val Glu Glu Ser Asn Phe Lys

195 200 205 act gtg gaa gct ctt gta gaa tct gtg tca gct gtt att gcc cat aac 672

Thr Val Glu Ala Leu Val Glu Ser Val Ser Ala Val Ile Ala His Asn

210 215 220 gag tat ttt caa aag ttt cca gat tcg cct ttg gtg gtg aag gtt ttg 720

Glu Tyr Phe Gin Lys Phe Pro Asp Ser Pro Leu Val Val Lys Val Leu

225 230 235 240 aaa tta aac gca atc aca gcc aca gaa ggt gtt ggt gta agc tgt att 768

Lys Leu Asn Ala Ile Thr Ala Thr Glu Gly Val Gly Val Ser Cys Ile

245 250 255 aga gag ccc agg gag att gcg atg gta aat att cca tat ctt tcc tcc 816

Arg Glu Pro Arg Glu Ile Ala Met Val Asn Ile Pro Tyr Leu Ser Ser

260 265 270 ata cat gaa tcg tct gat att aag ttc caa ttg tct tca tca caa aac 864 Ile His Glu Ser Ser Asp Ile Lys Phe Gin Leu Ser Ser Ser Gin Asn

275 280 285 act cct att gag ggt aaa aat aca tgg aaa aga gcg ttt tta gcg ttt 912

Thr Pro Ile Glu Gly Lys Asn Thr Trp Lys Arg Ala Phe Leu Ala Phe

290 295 300 ggt tca aac att ggg gac cgt ttc aaa cac att caa atg gcg ttg caa 960

Gly Ser Asn Ile Gly Asp Arg Phe Lys His Ile Gin Met Ala Leu Gin

305 310 315 320 tta tta tca agg gaa aaa acg gtt aaa tta egg aat att tcg tct att 1008

Leu Leu Ser Arg Glu Lys Thr Val Lys Leu Arg Asn Ile Ser Ser Ile

325 330 335 ttt gaa agt gaa cca atg tat ttc aaa gat caa acc cct ttc atg aat 1056

Phe Glu Ser Glu Pro Met Tyr Phe Lys Asp Gin Thr Pro Phe Met Asn

340 345 350 ggg tgt gtt gag gtg gag aca tta ctg acc cca agc gaa tta tta aaa 1104

Gly Cys Val Glu Val Glu Thr Leu Leu Thr Pro Ser Glu Leu Leu Lys

355 360 365 ttg tgt aaa aaa att gaa tat gaa gag ttg caa aga gtc aag cat ttt 1152 Leu Cys Lys Lys Ile Glu Tyr Glu Glu Leu Gin Arg Val Lys His Phe 370 375 380 gat aat ggt ccg aga aca ata gat ctg gat att gtt atg ttt ttg aat 1200

Asp Asn Gly Pro Arg Thr Ile Asp Leu Asp Ile Val Met Phe Leu Asn

385 390 395 400 agc gcc gga gaa gat att ata gta aat gaa ccg gat ttg aat ata ccg 1248

Ser Ala Gly Glu Asp Ile Ile Val Asn Glu Pro Asp Leu Asn Ile Pro

405 410 415 cat cct aga atg ctg gag agg act ttc gtt ctt gag ccg tta tgt gaa 1296

His Pro Arg Met Leu Glu Arg Thr Phe Val Leu Glu Pro Leu Cys Glu

420 425 430 tta ata tcc ccc gtt cac ctt cat cct gtg aca gcg gaa ccc att gta 1344

Leu Ile Ser Pro Val His Leu His Pro Val Thr Ala Glu Pro Ile Val

435 440 445 gac cat tta aaa cag tta tac gac aaa cag cat gat gaa gat acc tta 1392

Asp His Leu Lys Gin Leu Tyr Asp Lys Gin His Asp Glu Asp Thr Leu

450 455 460 tgg aaa tta gtt cca ttg cct tat cgt agt ggt gtg gag cct aga ttt 1440

Trp Lys Leu Val Pro Leu Pro Tyr Arg Ser Gly Val Glu Pro Arg Phe

465 470 475 480 ttg aaa ttc aag acc gct aca aaa ctt gac gaa ttt act gga gaa aca 1488

Leu Lys Phe Lys Thr Ala Thr Lys Leu Asp Glu Phe Thr Gly Glu Thr

485 490 495 aac aga att act gtt tca cct aca tat atc atg gct atc ttc aac gct 1536

Asn Arg Ile Thr Val Ser Pro Thr Tyr Ile Met Ala Ile Phe Asn Ala

500 505 510 aca cca gat tca ttt tcc gat gga ggt gag cat ttt gcg gac att gaa 1584

Thr Pro Asp Ser Phe Ser Asp Gly Gly Glu His Phe Ala Asp Ile Glu

515 520 525 agt caa ttg aat gat atc att aaa ttg tgt aaa gac gca tta tat ttg 1632

Ser Gin Leu Asn Asp Ile Ile Lys Leu Cys Lys Asp Ala Leu Tyr Leu

530 535 540 cat gag agc gtc atc atc gac gtt gga ggg tgt tct acc agg cct aac 1680

His Glu Ser Val Ile Ile Asp Val Gly Gly Cys Ser Thr Arg Pro Asn

545 550 555 560 tct att cag gcg tct gag gaa gaa gaa ata cgc agg tct atc cca tta 1728

Ser Ile Gin Ala Ser Glu Glu Glu Glu Ile Arg Arg Ser Ile Pro Leu

565 570 575 att aag gcc att aga gaa agc act gag tta ccg caa gat aaa gtc ata 1776 Ile Lys Ala Ile Arg Glu Ser Thr Glu Leu Pro Gin Asp Lys Val Ile

580 585 590 cta tcc att gat act tat cgt tcc aat gtc gct aaa gaa gcg att aaa 1824

Leu Ser Ile Asp Thr Tyr Arg Ser Asn Val Ala Lys Glu Ala Ile Lys

595 600 605 gtt gga gtg gat att att aat gat att tcg gga ggt tta ttt gac agc 1872 Val Gly Val Asp Ile Ile Asn Asp Ile Ser Gly Gly Leu Phe Asp Ser

610 615 620 aac atg ttt gcc gta att gca gag aac cca gaa att tgt tat att tta 1920

Asn Met Phe Ala Val Ile Ala Glu Asn Pro Glu Ile Cys Tyr Ile Leu

625 630 635 640 tca cac aca cgt ggt gat att tca acg atg aat agg ctg gcg cat tac 1968

Ser His Thr Arg Gly Asp Ile Ser Thr Met Asn Arg Leu Ala His Tyr

645 650 655 gaa aat ttt gca ttg ggt gat tct att cag caa gaa ttt gtt cat aat 2016

Glu Asn Phe Ala Leu Gly Asp Ser Ile Gin Gin Glu Phe Val His Asn

660 665 670 acc gac att cag cag cta gac gac ttg aaa gac aaa aca gtg tta atc 2064

Thr Asp Ile Gin Gin Leu Asp Asp Leu Lys Asp Lys Thr Val Leu Ile

675 680 685 agg aat gtt ggt caa gaa att ggc gaa agg tat atc aaa gcg att gat 2112

Arg Asn Val Gly Gin Glu Ile Gly Glu Arg Tyr Ile Lys Ala Ile Asp

690 695 700 aat gga gta aag cgc tgg caa att cta atc gac cct gga ctt ggt ttt 2160

Asn Gly Val Lys Arg Trp Gin Ile Leu Ile Asp Pro Gly Leu Gly Phe

705 710 715 720 gct aag acc tgg aag caa aac tta caa att att aga cat atc ccc att 2208

Ala Lys Thr Trp Lys Gin Asn Leu Gin Ile Ile Arg His Ile Pro Ile

725 730 735 tta aag aac tac tca ttc acc atg aac tca aac aat tcg caa gtg tat 2256

Leu Lys Asn Tyr Ser Phe Thr Met Asn Ser Asn Asn Ser Gin Val Tyr

740 745 750 gtt aac ctc aga aat atg ccc gtt tta ttg ggt cca tcg cgc aaa aaa 2304 Val Asn Leu Arg Asn Met Pro Val Leu Leu Gly Pro Ser Arg Lys Lys

755 760 765 ttc att gga cat atc aca aaa gat gtg gat gcg aag caa aga gac ttt 2352

Phe Ile Gly His Ile Thr Lys Asp Val Asp Ala Lys Gin Arg Asp Phe

770 775 780 gct act gga gcg gtg gta gcg tcg tgt att ggt ttc ggc agc gac atg 2400

Ala Thr Gly Ala Val Val Ala Ser Cys Ile Gly Phe Gly Ser Asp Met

785 790 795 800 gtt agg gtc cat gac gtt aaa aat tgt tcg aag agc att aaa tta gca 2448 Val Arg Val His Asp Val Lys Asn Cys Ser Lys Ser Ile Lys Leu Ala

805 810 815 gat gct att tat aaa ggt ttg gaa taa 2475

Asp Ala Ile Tyr Lys Gly Leu Glu

820

<210> 46

<211> 824

<212> PRT

<213> S . cerevisiae

<400> 46

Met Ser Lys Leu Phe Ser Thr Val Asn Ser Ala Arg His Ser Val Pro

1 5 10 15 Leu Gly Gly Met Arg Asp Tyr Val His Ile Lys Lys Leu Glu Met Asn 20 25 30

Thr Val Leu Gly Pro Asp Ser Trp Asn Gin Leu Met Pro Gin Lys Cys 35 40 45

Leu Leu Ser Leu Asp Met Gly Thr Asp Phe Ser Lys Ser Ala Ala Thr 50 55 60

Asp Asp Leu Lys Tyr Ser Leu Asn Tyr Ala Val Ile Ser Arg Asp Leu 65 70 75 80

Thr Asn Phe Val Ser Lys Lys Lys Asn Trp Gly Ser Val Ser Asn Leu 85 90 95

Ala Lys Ser Val Ser Gin Phe Val Met Asp Lys Tyr Ser Gly Val Glu

100 105 110

Cys Leu Asn Leu Glu Val Gin Ala Asp Thr Thr His Ile Arg Ser Asp

115 120 125

His Ile Ser Cys Ile Ile Gin Gin Glu Arg Gly Asn Pro Glu Ser Gin

130 135 140

Glu Phe Asp Val Val Arg Ile Ser Glu Leu Lys Met Leu Thr Leu Ile 145 150 155 160

Gly Val Phe Thr Phe Glu Arg Leu Lys Lys Gin Tyr Val Thr Leu Asp 165 170 175 Ile Lys Leu Pro Trp Pro Lys Lys Ala Glu Leu Pro Pro Pro Val Gin

180 185 190

Ser Ile Ile Asp Asn Val Val Lys Phe Val Glu Glu Ser Asn Phe Lys 195 200 205

Thr Val Glu Ala Leu Val Glu Ser Val Ser Ala Val Ile Ala His Asn

210 215 220

Glu Tyr Phe Gin Lys Phe Pro Asp Ser Pro Leu Val Val Lys Val Leu 225 230 235 240

Lys Leu Asn Ala Ile Thr Ala Thr Glu Gly Val Gly Val Ser Cys Ile 245 250 255

Arg Glu Pro Arg Glu Ile Ala Met Val Asn Ile Pro Tyr Leu Ser Ser

260 265 270 Ile His Glu Ser Ser Asp Ile Lys Phe Gin Leu Ser Ser Ser Gin Asn

275 280 285

Thr Pro Ile Glu Gly Lys Asn Thr Trp Lys Arg Ala Phe Leu Ala Phe

290 295 300

Gly Ser Asn Ile Gly Asp Arg Phe Lys His Ile Gin Met Ala Leu Gin 305 310 315 320

Leu Leu Ser Arg Glu Lys Thr Val Lys Leu Arg Asn Ile Ser Ser Ile 325 330 335

Phe Glu Ser Glu Pro Met Tyr Phe Lys Asp Gin Thr Pro Phe Met Asn

340 345 350

Gly Cys Val Glu Val Glu Thr Leu Leu Thr Pro Ser Glu Leu Leu Lys 355 360 365

Leu Cys Lys Lys Ile Glu Tyr Glu Glu Leu Gin Arg Val Lys His Phe

370 375 380

Asp Asn Gly Pro Arg Thr Ile Asp Leu Asp Ile Val Met Phe Leu Asn 385 390 395 400

Ser Ala Gly Glu Asp Ile Ile Val Asn Glu Pro Asp Leu Asn Ile Pro 405 410 415

His Pro Arg Met Leu Glu Arg Thr Phe Val Leu Glu Pro Leu Cys Glu

420 425 430

Leu Ile Ser Pro Val His Leu His Pro Val Thr Ala Glu Pro Ile Val

435 440 445

Asp His Leu Lys Gin Leu Tyr Asp Lys Gin His Asp Glu Asp Thr Leu

450 455 460

Trp Lys Leu Val Pro Leu Pro Tyr Arg Ser Gly Val Glu Pro Arg Phe 465 470 475 480

Leu Lys Phe Lys Thr Ala Thr Lys Leu Asp Glu Phe Thr Gly Glu Thr 485 490 495

Asn Arg Ile Thr Val Ser Pro Thr Tyr Ile Met Ala Ile Phe Asn Ala

500 505 510

Thr Pro Asp Ser Phe Ser Asp Gly Gly Glu His Phe Ala Asp Ile Glu 515 520 525

Ser Gin Leu Asn Asp Ile Ile Lys Leu Cys Lys Asp Ala Leu Tyr Leu

530 535 540

His Glu Ser Val Ile Ile Asp Val Gly Gly Cys Ser Thr Arg Pro Asn 545 550 555 560

Ser Ile Gin Ala Ser Glu Glu Glu Glu Ile Arg Arg Ser Ile Pro Leu

565 570 575 Ile Lys Ala Ile Arg Glu Ser Thr Glu Leu Pro Gin Asp Lys Val Ile

580 585 590

Leu Ser Ile Asp Thr Tyr Arg Ser Asn Val Ala Lys Glu Ala Ile Lys 595 600 605 Val Gly Val Asp Ile Ile Asn Asp Ile Ser Gly Gly Leu Phe Asp Ser

610 615 620

Asn Met Phe Ala Val Ile Ala Glu Asn Pro Glu Ile Cys Tyr Ile Leu 625 630 635 640

Ser His Thr Arg Gly Asp Ile Ser Thr Met Asn Arg Leu Ala His Tyr

645 650 655

Glu Asn Phe Ala Leu Gly Asp Ser Ile Gin Gin Glu Phe Val His Asn 660 665 670

Thr Asp Ile Gin Gin Leu Asp Asp Leu Lys Asp Lys Thr Val Leu Ile 675 680 685

Arg Asn Val Gly Gin Glu Ile Gly Glu Arg Tyr Ile Lys Ala Ile Asp

690 695 700

Asn Gly Val Lys Arg Trp Gin Ile Leu Ile Asp Pro Gly Leu Gly Phe 705 710 715 720

Ala Lys Thr Trp Lys Gin Asn Leu Gin Ile Ile Arg His Ile Pro Ile

725 730 735

Leu Lys Asn Tyr Ser Phe Thr Met Asn Ser Asn Asn Ser Gin Val Tyr 740 745 750 Val Asn Leu Arg Asn Met Pro Val Leu Leu Gly Pro Ser Arg Lys Lys 755 760 765 Phe Ile Gly His Ile Thr Lys Asp Val Asp Ala Lys Gin Arg Asp Phe

770 775 780

Ala Thr Gly Ala Val Val Ala Ser Cys Ile Gly Phe Gly Ser Asp Met 785 790 795 800 Val Arg Val His Asp Val Lys Asn Cys Ser Lys Ser Ile Lys Leu Ala

805 810 815

Asp Ala Ile Tyr Lys Gly Leu Glu 820

<210> 47

<211> 669

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . ( 669)

<223> S . cerevisiae essential gene MED7 YOL135C SGDID : S000005495

<400> 47 atg tcc aat gat cct ggt aat gag gtt agc tcc ttg tac cca cct cca 48 Met Ser Asn Asp Pro Gly Asn Glu Val Ser Ser Leu Tyr Pro Pro Pro 1 5 10 15 ccc cca tac gtg aag ttt ttc aca cag tcc aat ctg gaa aaa tta cca 96

Pro Pro Tyr Val Lys Phe Phe Thr Gin Ser Asn Leu Glu Lys Leu Pro

20 25 30 aaa tac aaa gaa aag aag gcc gca agt gcg aag cag acg gct cca aat 144

Lys Tyr Lys Glu Lys Lys Ala Ala Ser Ala Lys Gin Thr Ala Pro Asn

35 40 45 aac agt aac ggc ggc agt gaa gag gag att aca tgt gcg cta gat tac 192

Asn Ser Asn Gly Gly Ser Glu Glu Glu Ile Thr Cys Ala Leu Asp Tyr

50 55 60 cta atc cca ccc ccc atg ccc aaa aat cag cag tac cga gcg ttt ggg 240

Leu Ile Pro Pro Pro Met Pro Lys Asn Gin Gin Tyr Arg Ala Phe Gly

65 70 75 80 agt ata tgg cag gtg aag gac caa ctg ccc gat tta gag agc atg gga 288

Ser Ile Trp Gin Val Lys Asp Gin Leu Pro Asp Leu Glu Ser Met Gly

85 90 95 ctc acc caa ctt tat aag aaa tcc aca gaa aac gaa agc aca aat tat 336

Leu Thr Gin Leu Tyr Lys Lys Ser Thr Glu Asn Glu Ser Thr Asn Tyr

100 105 110 caa tac aaa atc caa gag cta cgc aaa cta ttg aaa tcc ctc ctg cta 384

Gin Tyr Lys Ile Gin Glu Leu Arg Lys Leu Leu Lys Ser Leu Leu Leu

115 120 125 aat tac ttg gaa ctc atc ggc gta ttg agc ata aat ccg gat atg tat 432

Asn Tyr Leu Glu Leu Ile Gly Val Leu Ser Ile Asn Pro Asp Met Tyr

130 135 140 gag cgc aaa gtg gag aac ata agg acc att ctt gtc aat att cac cac 480

Glu Arg Lys Val Glu Asn Ile Arg Thr Ile Leu Val Asn Ile His His

145 150 155 160 cta ctt aat gag tac aga ccc cat cag tcc cga gag tcc ctt att atg 528

Leu Leu Asn Glu Tyr Arg Pro His Gin Ser Arg Glu Ser Leu Ile Met

165 170 175 cta ttg gaa gaa caa tta gaa tac aag cgc ggg gaa att cgc gag atc 576

Leu Leu Glu Glu Gin Leu Glu Tyr Lys Arg Gly Glu Ile Arg Glu Ile

180 185 190 gag caa gta tgc aaa cag gta cac gac aaa ctc acc agc ata cag gac 624

Glu Gin Val Cys Lys Gin Val His Asp Lys Leu Thr Ser Ile Gin Asp

195 200 205 acc tta aga acc gga tca caa tct cct cca tcg tcg tcc caa tga 669

Thr Leu Arg Thr Gly Ser Gin Ser Pro Pro Ser Ser Ser Gin

210 215 220

<210> 48

<211> 222

<212> PRT

<213> S . cerevisiae

<400> 48

Met Ser Asn Asp Pro Gly Asn Glu Val Ser Ser Leu Tyr Pro Pro Pro 1 5 10 15

Pro Pro Tyr Val Lys Phe Phe Thr Gin Ser Asn Leu Glu Lys Leu Pro 20 25 30

Lys Tyr Lys Glu Lys Lys Ala Ala Ser Ala Lys Gin Thr Ala Pro Asn 35 40 45

Asn Ser Asn Gly Gly Ser Glu Glu Glu Ile Thr Cys Ala Leu Asp Tyr 50 55 60

Leu Ile Pro Pro Pro Met Pro Lys Asn Gin Gin Tyr Arg Ala Phe Gly 65 70 75 80

Ser Ile Trp Gin Val Lys Asp Gin Leu Pro Asp Leu Glu Ser Met Gly 85 90 95

Leu Thr Gin Leu Tyr Lys Lys Ser Thr Glu Asn Glu Ser Thr Asn Tyr 100 105 110

Gin Tyr Lys Ile Gin Glu Leu Arg Lys Leu Leu Lys Ser Leu Leu Leu 115 120 125

Asn Tyr Leu Glu Leu Ile Gly Val Leu Ser Ile Asn Pro Asp Met Tyr 130 135 140 Glu Arg Lys Val Glu Asn Ile Arg Thr Ile Leu Val Asn Ile His His 145 150 155 160

Leu Leu Asn Glu Tyr Arg Pro His Gin Ser Arg Glu Ser Leu Ile Met

165 170 175

Leu Leu Glu Glu Gin Leu Glu Tyr Lys Arg Gly Glu Ile Arg Glu Ile 180 185 190

Glu Gin Val Cys Lys Gin Val His Asp Lys Leu Thr Ser Ile Gin Asp 195 200 205

Thr Leu Arg Thr Gly Ser Gin Ser Pro Pro Ser Ser Ser Gin

210 215 220

<210> 49

<211> 723

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . (723)

<223> S . cerevisiae essential gene RRP40 YOL142W SGD1D : S000005502

<400> 49 atg tct acg ttc ata ttc cct ggt gat agc ttt cct gta gac cct act 48 Met Ser Thr Phe Ile Phe Pro Gly Asp Ser Phe Pro Val Asp Pro Thr 1 5 10 15 aca cct gtt aaa ctg ggc cct ggc ata tat tgt gac ccc aat act caa 96 Thr Pro Val Lys Leu Gly Pro Gly Ile Tyr Cys Asp Pro Asn Thr Gin 20 25 30 gaa ata cga cct gtt aat aca ggt gtt ttg cat gtt tcc gct aag ggt 144 Glu Ile Arg Pro Val Asn Thr Gly Val Leu His Val Ser Ala Lys Gly 35 40 45 aag agt ggt gtt cag acc gca tat ata gac tat tct agt aag aga tac 192 Lys Ser Gly Val Gin Thr Ala Tyr Ile Asp Tyr Ser Ser Lys Arg Tyr 50 55 60 att cca tct gta aac gat ttt gta atc ggt gtc att ata ggg aca ttt 240 Ile Pro Ser Val Asn Asp Phe Val Ile Gly Val Ile Ile Gly Thr Phe 65 70 75 80 tca gat agc tat aag gtt tcg ttg caa aat ttc tec tcc agt gtt tca 288 Ser Asp Ser Tyr Lys Val Ser Leu Gin Asn Phe Ser Ser Ser Val Ser 85 90 95 cta tcg tat atg gct ttt ccc aat gct tca aag aaa aac agg cca act 336

Leu Ser Tyr Met Ala Phe Pro Asn Ala Ser Lys Lys Asn Arg Pro Thr 100 105 110 ttg caa gtg gga gat cta gtg tat gcg aga gtt tgt acc gca gaa aag 384 Leu Gin Val Gly Asp Leu Val Tyr Ala Arg Val Cys Thr Ala Glu Lys

115 120 125 gaa cta gaa gcc gaa ata gaa tgt ttt gac tca act aca gga cgc gat 432

Glu Leu Glu Ala Glu Ile Glu Cys Phe Asp Ser Thr Thr Gly Arg Asp

130 135 140 gct ggt ttc ggg ata ttg gaa gat ggt atg atc att gac gtg aat ttg 480

Ala Gly Phe Gly Ile Leu Glu Asp Gly Met Ile Ile Asp Val Asn Leu

145 150 155 160 aat ttc gca cgc cag ttg ctt ttc aat aat gac ttc ccg tta tta aaa 528

Asn Phe Ala Arg Gin Leu Leu Phe Asn Asn Asp Phe Pro Leu Leu Lys

165 170 175 gtg ttg gct gca cat acc aag ttt gaa gtc gcc att ggt ctc aat ggg 576 Val Leu Ala Ala His Thr Lys Phe Glu Val Ala Ile Gly Leu Asn Gly

180 185 190 aag atc tgg gtt aag tgc gag gaa tta tct aac act tta gct tgt tat 624

Lys Ile Trp Val Lys Cys Glu Glu Leu Ser Asn Thr Leu Ala Cys Tyr

195 200 205 aga acc ata atg gag tgt tgt caa aaa aac gac acg gca gcg ttc aag 672

Arg Thr Ile Met Glu Cys Cys Gin Lys Asn Asp Thr Ala Ala Phe Lys

210 215 220 gat ata gca aaa aga cag ttt aaa gaa ata ctt acg gtc aag gag gag 720

Asp Ile Ala Lys Arg Gin Phe Lys Glu Ile Leu Thr Val Lys Glu Glu

225 230 235 240 tag 723

<210> 50

<211> 240

<212> PRT

<213> S . cerevisiae

<400> 50

Met Ser Thr Phe Ile Phe Pro Gly Asp Ser Phe Pro Val Asp Pro Thr

1 5 10 15

Thr Pro Val Lys Leu Gly Pro Gly Ile Tyr Cys Asp Pro Asn Thr Gin 20 25 30

Glu Ile Arg Pro Val Asn Thr Gly Val Leu His Val Ser Ala Lys Gly 35 40 45

Lys Ser Gly Val Gin Thr Ala Tyr Ile Asp Tyr Ser Ser Lys Arg Tyr 50 55 60 Ile Pro Ser Val Asn Asp Phe Val Ile Gly Val Ile Ile Gly Thr Phe 65 70 75 80

Ser Asp Ser Tyr Lys Val Ser Leu Gin Asn Phe Ser Ser Ser Val Ser 85 90 95 Leu Ser Tyr Met Ala Phe Pro Asn Ala Ser Lys Lys Asn Arg Pro Thr 100 105 110

Leu Gin Val Gly Asp Leu Val Tyr Ala Arg Val Cys Thr Ala Glu Lys 115 120 125

Glu Leu Glu Ala Glu Ile Glu Cys Phe Asp Ser Thr Thr Gly Arg Asp

130 135 140

Ala Gly Phe Gly Ile Leu Glu Asp Gly Met Ile Ile Asp Val Asn Leu 145 150 155 160

Asn Phe Ala Arg Gin Leu Leu Phe Asn Asn Asp Phe Pro Leu Leu Lys

165 170 175 Val Leu Ala Ala His Thr Lys Phe Glu Val Ala Ile Gly Leu Asn Gly 180 185 190

Lys Ile Trp Val Lys Cys Glu Glu Leu Ser Asn Thr Leu Ala Cys Tyr 195 200 205

Arg Thr Ile Met Glu Cys Cys Gin Lys Asn Asp Thr Ala Ala Phe Lys

210 215 220

Asp Ile Ala Lys Arg Gin Phe Lys Glu Ile Leu Thr Val Lys Glu Glu 225 230 235 240

<210> 51

<211> 1455

<212> DNA

<213> S . cerevisiea

<220>

<221> CDS

<222> (1) . . (1455)

<223> S . cerevisiae essential gene NOP8 YOL144W SGD1D : S000005504

<400> 51 atg gat agt gta att caa aaa aga att ttt gtc gga aat att ttc cat 48

Met Asp Ser Val Ile Gin Lys Arg Ile Phe Val Gly Asn Ile Phe His

1 5 10 15 aat gca gat gat tgt tat tca gaa cta cta gat cga ttt gga aaa ttt 96

Asn Ala Asp Asp Cys Tyr Ser Glu Leu Leu Asp Arg Phe Gly Lys Phe

20 25 30 ggt gat tgc caa gat ttt cag ttt gaa aaa cac aac cac ttt gca ttt 144

Gly Asp Cys Gin Asp Phe Gin Phe Glu Lys His Asn His Phe Ala Phe

35 40 45 atc gat ata agg ttt aat gat gag gca gat ttt aac aag cta aga aag 192 Ile Asp Ile Arg Phe Asn Asp Glu Ala Asp Phe Asn Lys Leu Arg Lys 50 55 60 agc ttt aat aat gtt aag ttt aaa ggg aac att ttg aag gtt gat gaa 240

Ser Phe Asn Asn Val Lys Phe Lys Gly Asn Ile Leu Lys Val Asp Glu

65 70 75 80 gca aag ccg aat tgg gaa agc acc tgg gcg gta caa cat gca aag gac 288

Ala Lys Pro Asn Trp Glu Ser Thr Trp Ala Val Gin His Ala Lys Asp

85 90 95 ctt aaa gaa gat att ata cta aat gct aaa atg aaa aaa aag aat tgg 336

Leu Lys Glu Asp Ile Ile Leu Asn Ala Lys Met Lys Lys Lys Asn Trp

100 105 110 caa cat tat aag aaa atg gaa aat gta gcg aaa agt tgg aaa gat cat 384

Gin His Tyr Lys Lys Met Glu Asn Val Ala Lys Ser Trp Lys Asp His

115 120 125 aag gaa gtt att gct ggc aga atg agg gag gcg ccg aga aaa agg agt 432

Lys Glu Val Ile Ala Gly Arg Met Arg Glu Ala Pro Arg Lys Arg Ser

130 135 140 caa ttg agg aac att aca ttc agg atc aat gtc aat ggt tca ttg aaa 480

Gin Leu Arg Asn Ile Thr Phe Arg Ile Asn Val Asn Gly Ser Leu Lys

145 150 155 160 gtt tac aaa tgc tat aag act aaa tta tgg ggt tat gaa agg aac aaa 528 Val Tyr Lys Cys Tyr Lys Thr Lys Leu Trp Gly Tyr Glu Arg Asn Lys

165 170 175 gaa ttg aat gac ctt gtg tac aag ttt aca aat aat ttc tgg aaa aac 576

Glu Leu Asn Asp Leu Val Tyr Lys Phe Thr Asn Asn Phe Trp Lys Asn

180 185 190 gga tac aac cac att gtc gat aga ttg gat tac agt cgt gct gtt aaa 624

Gly Tyr Asn His Ile Val Asp Arg Leu Asp Tyr Ser Arg Ala Val Lys

195 200 205 act gtt cga ttc aaa aat ggg ctg aaa cag tta aca gtg tca aaa gat 672

Thr Val Arg Phe Lys Asn Gly Leu Lys Gin Leu Thr Val Ser Lys Asp

210 215 220 gaa aac gtt tgc agt gga gag atg gac agc gat gag aat atg tca gaa 720

Glu Asn Val Cys Ser Gly Glu Met Asp Ser Asp Glu Asn Met Ser Glu

225 230 235 240 gag gag aag gag aaa aat aat gtt att ctt aat gac cta tta aag gac 768

Glu Glu Lys Glu Lys Asn Asn Val Ile Leu Asn Asp Leu Leu Lys Asp

245 250 255 ttt gac ttc gat aag cca atg acg ttg aat gat tcg gac gag gaa ctt 816

Phe Asp Phe Asp Lys Pro Met Thr Leu Asn Asp Ser Asp Glu Glu Leu

260 265 270 ctg aca gag caa cgt aaa gga gag gaa gaa gaa gaa gaa gaa gaa gaa 864

Leu Thr Glu Gin Arg Lys Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu

275 280 285 aaa gaa gta aac gct ccc gaa tac gag aat gtt aat aag acg aag gac 912

Lys Glu Val Asn Ala Pro Glu Tyr Glu Asn Val Asn Lys Thr Lys Asp

290 295 300 cag agc acc cta cca caa gaa aaa cca gag gaa agg aaa gaa cag gac 960 Gin Ser Thr Leu Pro Gin Glu Lys Pro Glu Glu Arg Lys Glu Gin Asp 305 310 315 320 gag ggc gac ggg cag gag gat aat gaa ttt att ccg act ttc acc aag 1008

Glu Gly Asp Gly Gin Glu Asp Asn Glu Phe Ile Pro Thr Phe Thr Lys

325 330 335 gaa att ggt caa ggt aca ata agc aac act gag acg tta aga aac cta 1056

Glu Ile Gly Gin Gly Thr Ile Ser Asn Thr Glu Thr Leu Arg Asn Leu

340 345 350 ttc aac ccc aac gag gca gaa cct gta tca caa ttc aaa ttg att gaa 1104

Phe Asn Pro Asn Glu Ala Glu Pro Val Ser Gin Phe Lys Leu Ile Glu

355 360 365 gat tct gat aat gat att gat cat gcg aaa gat gtt gat gtg aac caa 1152

Asp Ser Asp Asn Asp Ile Asp His Ala Lys Asp Val Asp Val Asn Gin

370 375 380 ctg gag gaa gaa gta agt aaa tca tct gac acc cta ggt ttg aca tcg 1200

Leu Glu Glu Glu Val Ser Lys Ser Ser Asp Thr Leu Gly Leu Thr Ser

385 390 395 400 gct ccc gta cca cat gta tct aga gat aaa gat aac aaa aat ttc ttg 1248

Ala Pro Val Pro His Val Ser Arg Asp Lys Asp Asn Lys Asn Phe Leu

405 410 415 ttt ttc cct cat ttg caa tcg cca ttt tta gta gga caa aca cag ttg 1296

Phe Phe Pro His Leu Gin Ser Pro Phe Leu Val Gly Gin Thr Gin Leu

420 425 430 agt aaa gta agg gct cct gga aga gaa aca atg cta tcg aac tgg gat 1344

Ser Lys Val Arg Ala Pro Gly Arg Glu Thr Met Leu Ser Asn Trp Asp

435 440 445 gag gag ttc tgg gct aac aga ggt aat tgg acc cgt gat atg agg cgc 1392

Glu Glu Phe Trp Ala Asn Arg Gly Asn Trp Thr Arg Asp Met Arg Arg

450 455 460 aaa atg aaa gat gca ttg aag cac cgt aag agg aaa caa tca aag agc 1440

Lys Met Lys Asp Ala Leu Lys His Arg Lys Arg Lys Gin Ser Lys Ser

465 470 475 480 ggg ctt ctt cta tag 1455 Gly Leu Leu Leu

<210> 52

<211> 484

<212> PRT

<213> S . cerevisiea

<400> 52

Met Asp Ser Val Ile Gin Lys Arg Ile Phe Val Gly Asn Ile Phe His

1 5 10 15

Asn Ala Asp Asp Cys Tyr Ser Glu Leu Leu Asp Arg Phe Gly Lys Phe 20 25 30 Gly Asp Cys Gin Asp Phe Gin Phe Glu Lys His Asn His Phe Ala Phe 35 40 45 Ile Asp Ile Arg Phe Asn Asp Glu Ala Asp Phe Asn Lys Leu Arg Lys

50 55 60

Ser Phe Asn Asn Val Lys Phe Lys Gly Asn Ile Leu Lys Val Asp Glu 65 70 75 80

Ala Lys Pro Asn Trp Glu Ser Thr Trp Ala Val Gin His Ala Lys Asp 85 90 95

Leu Lys Glu Asp Ile Ile Leu Asn Ala Lys Met Lys Lys Lys Asn Trp

100 105 110

Gin His Tyr Lys Lys Met Glu Asn Val Ala Lys Ser Trp Lys Asp His 115 120 125

Lys Glu Val Ile Ala Gly Arg Met Arg Glu Ala Pro Arg Lys Arg Ser

130 135 140

Gin Leu Arg Asn Ile Thr Phe Arg Ile Asn Val Asn Gly Ser Leu Lys 145 150 155 160 Val Tyr Lys Cys Tyr Lys Thr Lys Leu Trp Gly Tyr Glu Arg Asn Lys 165 170 175

Glu Leu Asn Asp Leu Val Tyr Lys Phe Thr Asn Asn Phe Trp Lys Asn

180 185 190

Gly Tyr Asn His Ile Val Asp Arg Leu Asp Tyr Ser Arg Ala Val Lys 195 200 205

Thr Val Arg Phe Lys Asn Gly Leu Lys Gin Leu Thr Val Ser Lys Asp

210 215 220

Glu Asn Val Cys Ser Gly Glu Met Asp Ser Asp Glu Asn Met Ser Glu 225 230 235 240

Glu Glu Lys Glu Lys Asn Asn Val Ile Leu Asn Asp Leu Leu Lys Asp 245 250 255

Phe Asp Phe Asp Lys Pro Met Thr Leu Asn Asp Ser Asp Glu Glu Leu

260 265 270

Leu Thr Glu Gin Arg Lys Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu 275 280 285 Lys Glu Val Asn Ala Pro Glu Tyr Glu Asn Val Asn Lys Thr Lys Asp

290 295 300

Gin Ser Thr Leu Pro Gin Glu Lys Pro Glu Glu Arg Lys Glu Gin Asp 305 310 315 320

Glu Gly Asp Gly Gin Glu Asp Asn Glu Phe Ile Pro Thr Phe Thr Lys 325 330 335

Glu Ile Gly Gin Gly Thr Ile Ser Asn Thr Glu Thr Leu Arg Asn Leu

340 345 350

Phe Asn Pro Asn Glu Ala Glu Pro Val Ser Gin Phe Lys Leu Ile Glu 355 360 365

Asp Ser Asp Asn Asp Ile Asp His Ala Lys Asp Val Asp Val Asn Gin

370 375 380

Leu Glu Glu Glu Val Ser Lys Ser Ser Asp Thr Leu Gly Leu Thr Ser 385 390 395 400

Ala Pro Val Pro His Val Ser Arg Asp Lys Asp Asn Lys Asn Phe Leu 405 410 415

Phe Phe Pro His Leu Gin Ser Pro Phe Leu Val Gly Gin Thr Gin Leu

420 425 430

Ser Lys Val Arg Ala Pro Gly Arg Glu Thr Met Leu Ser Asn Trp Asp

435 440 445

Glu Glu Phe Trp Ala Asn Arg Gly Asn Trp Thr Arg Asp Met Arg Arg

450 455 460

Lys Met Lys Asp Ala Leu Lys His Arg Lys Arg Lys Gin Ser Lys Ser 465 470 475 480

Gly Leu Leu Leu

<210> 53

<211> 1665

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . (1665) <223> S . cerevisiae essential gene PGI1 YBR196C SGD1D : S000000400

<400> 53 atg tcc aat aac tca ttc act aac ttc aaa ctg gcc act gaa ttg cca 48

Met Ser Asn Asn Ser Phe Thr Asn Phe Lys Leu Ala Thr Glu Leu Pro 1 5 10 15 gcc tgg tct aag ttg caa aaa att tat gaa tct caa ggt aag act ttg 96

Ala Trp Ser Lys Leu Gin Lys Ile Tyr Glu Ser Gin Gly Lys Thr Leu

20 25 30 tct gtc aag caa gaa ttc caa aaa gat gcc aag cgt ttt gaa aaa ttg 144

Ser Val Lys Gin Glu Phe Gin Lys Asp Ala Lys Arg Phe Glu Lys Leu

35 40 45 aac aag act ttc acc aac tat gat ggt tcc aaa atc ttg ttc gac tac 192

Asn Lys Thr Phe Thr Asn Tyr Asp Gly Ser Lys Ile Leu Phe Asp Tyr

50 55 60 tca aag aac ttg gtc aac gat gaa atc att gct gca ttg att gaa ctg 240

Ser Lys Asn Leu Val Asn Asp Glu Ile Ile Ala Ala Leu Ile Glu Leu

65 70 75 80 gcc aag gag gct aac gtc acc ggt ttg aga gat gct atg ttc aaa ggt 288

Ala Lys Glu Ala Asn Val Thr Gly Leu Arg Asp Ala Met Phe Lys Gly

85 90 95 gaa cac atc aac tcc act gaa gat cgt gct gtc tac cac gtc gca ttg 336

Glu His Ile Asn Ser Thr Glu Asp Arg Ala Val Tyr His Val Ala Leu

100 105 110 aga aac aga gct aac aag cca atg tac gtt gat ggt gtc aac gtt gct 384

Arg Asn Arg Ala Asn Lys Pro Met Tyr Val Asp Gly Val Asn Val Ala

115 120 125 cca gaa gtc gac tct gtc ttg aag cac atg aag gag ttc tct gaa caa 432

Pro Glu Val Asp Ser Val Leu Lys His Met Lys Glu Phe Ser Glu Gin

130 135 140 gtt cgt tct ggt gaa tgg aag ggt tat acc ggt aag aag atc acc gat 480 Val Arg Ser Gly Glu Trp Lys Gly Tyr Thr Gly Lys Lys Ile Thr Asp

145 150 155 160 gtt gtt aac atc ggt att ggt ggt tcc gat ttg ggt cca gtc atg gtc 528 Val Val Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Val Met Val

165 170 175 act gag gct ttg aag cac tac gct ggt gtc ttg gat gtc cac ttc gtt 576

Thr Glu Ala Leu Lys His Tyr Ala Gly Val Leu Asp Val His Phe Val

180 185 190 tcc aac att gac ggt act cac att gct gaa acc ttg aag gtt gtt gac 624

Ser Asn Ile Asp Gly Thr His Ile Ala Glu Thr Leu Lys Val Val Asp

195 200 205 cca gaa act act ttg ttt ttg att gct tcc aag act ttc act acc gct 672

Pro Glu Thr Thr Leu Phe Leu Ile Ala Ser Lys Thr Phe Thr Thr Ala

210 215 220 gaa act atc act aac gct aac act gcc aag aac tgg ttc ttg tcg aag 720

Glu Thr Ile Thr Asn Ala Asn Thr Ala Lys Asn Trp Phe Leu Ser Lys

225 230 235 240 aca ggt aat gat cca tct cac att gct aag cat ttc gct gct ttg tec 768 Thr Gly Asn Asp Pro Ser His Ile Ala Lys His Phe Ala Ala Leu Ser 245 250 255 act aac gaa acc gaa gtt gcc aag ttc ggt att gac acc aaa aac atg 816

Thr Asn Glu Thr Glu Val Ala Lys Phe Gly Ile Asp Thr Lys Asn Met

260 265 270 ttt ggt ttc gaa agt tgg gtc ggt ggt cgt tac tct gtc tgg tcg gct 864

Phe Gly Phe Glu Ser Trp Val Gly Gly Arg Tyr Ser Val Trp Ser Ala

275 280 285 att ggt ttg tct gtt gcc ttg tac att ggc tat gac aac ttt gag gct 912 Ile Gly Leu Ser Val Ala Leu Tyr Ile Gly Tyr Asp Asn Phe Glu Ala

290 295 300 ttc ttg aag ggt gct gaa gcc gtc gac aac cac ttc acc caa acc cca 960

Phe Leu Lys Gly Ala Glu Ala Val Asp Asn His Phe Thr Gin Thr Pro

305 310 315 320 ttg gaa gac aac att cca ttg ttg ggt ggt ttg ttg tct gtc tgg tac 1008

Leu Glu Asp Asn Ile Pro Leu Leu Gly Gly Leu Leu Ser Val Trp Tyr

325 330 335 aac aac ttc ttt ggt gct caa acc cat ttg gtt gct cca ttc gac caa 1056

Asn Asn Phe Phe Gly Ala Gin Thr His Leu Val Ala Pro Phe Asp Gin

340 345 350 tac ttg cac aga ttc cca gcc tac ttg caa caa ttg tca atg gaa tct 1104

Tyr Leu His Arg Phe Pro Ala Tyr Leu Gin Gin Leu Ser Met Glu Ser

355 360 365 aac ggt aag tct gtt acc aga ggt aac gtg ttt act gac tac tct act 1152

Asn Gly Lys Ser Val Thr Arg Gly Asn Val Phe Thr Asp Tyr Ser Thr

370 375 380 ggt tct atc ttg ttt ggt gaa cca gct acc aac gct caa cac tct ttc 1200

Gly Ser Ile Leu Phe Gly Glu Pro Ala Thr Asn Ala Gin His Ser Phe

385 390 395 400 ttc caa ttg gtt cac caa ggt acc aag ttg att cca tct gat ttc atc 1248

Phe Gin Leu Val His Gin Gly Thr Lys Leu Ile Pro Ser Asp Phe Ile

405 410 415 tta gct gct caa tct cat aac cca att gag aac aaa tta cat caa aag 1296

Leu Ala Ala Gin Ser His Asn Pro Ile Glu Asn Lys Leu His Gin Lys

420 425 430 atg ttg gct tca aac ttc ttt gct caa gct gaa gct tta atg gtt ggt 1344

Met Leu Ala Ser Asn Phe Phe Ala Gin Ala Glu Ala Leu Met Val Gly

435 440 445 aag gat gaa gaa caa gtt aag gct gaa ggt gcc act ggt ggt ttg gtc 1392

Lys Asp Glu Glu Gin Val Lys Ala Glu Gly Ala Thr Gly Gly Leu Val

450 455 460 cca cac aag gtc ttc tca ggt aac aga cca act acc tct atc ttg gct 1440

Pro His Lys Val Phe Ser Gly Asn Arg Pro Thr Thr Ser Ile Leu Ala

465 470 475 480 caa aag att act cca gct act ttg ggt gct ttg att gcc tac tac gaa 1488

Gin Lys Ile Thr Pro Ala Thr Leu Gly Ala Leu Ile Ala Tyr Tyr Glu

485 490 495 cat gtt act ttc act gaa ggt gcc att tgg aat atc aac tct ttc gac 1536

His Val Thr Phe Thr Glu Gly Ala Ile Trp Asn Ile Asn Ser Phe Asp 500 505 510 caa tgg ggt gtt gaa ttg ggt aaa gtc ttg gct aaa gtc atc ggc aag 1584

Gin Trp Gly Val Glu Leu Gly Lys Val Leu Ala Lys Val Ile Gly Lys

515 520 525 gaa ttg gac aac tec tec acc att tct acc cac gat gct tct acc aac 1632 Glu Leu Asp Asn Ser Ser Thr Ile Ser Thr His Asp Ala Ser Thr Asn 530 535 540 ggt tta atc aat caa ttc aag gaa tgg atg tga 1665

Gly Leu Ile Asn Gin Phe Lys Glu Trp Met 545 550

<210> 54

<211> 554

<212> PRT

<213> S . cerevisiae

<400> 54

Met Ser Asn Asn Ser Phe Thr Asn Phe Lys Leu Ala Thr Glu Leu Pro 1 5 10 15

Ala Trp Ser Lys Leu Gin Lys Ile Tyr Glu Ser Gin Gly Lys Thr Leu 20 25 30

Ser Val Lys Gin Glu Phe Gin Lys Asp Ala Lys Arg Phe Glu Lys Leu 35 40 45

Asn Lys Thr Phe Thr Asn Tyr Asp Gly Ser Lys Ile Leu Phe Asp Tyr 50 55 60

Ser Lys Asn Leu Val Asn Asp Glu Ile Ile Ala Ala Leu Ile Glu Leu 65 70 75 80

Ala Lys Glu Ala Asn Val Thr Gly Leu Arg Asp Ala Met Phe Lys Gly 85 90 95

Glu His Ile Asn Ser Thr Glu Asp Arg Ala Val Tyr His Val Ala Leu 100 105 110

Arg Asn Arg Ala Asn Lys Pro Met Tyr Val Asp Gly Val Asn Val Ala 115 120 125

Pro Glu Val Asp Ser Val Leu Lys His Met Lys Glu Phe Ser Glu Gin 130 135 140 Val Arg Ser Gly Glu Trp Lys Gly Tyr Thr Gly Lys Lys Ile Thr Asp 145 150 155 160 Val Val Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Val Met Val 165 170 175

Thr Glu Ala Leu Lys His Tyr Ala Gly Val Leu Asp Val His Phe Val

180 185 190

Ser Asn Ile Asp Gly Thr His Ile Ala Glu Thr Leu Lys Val Val Asp 195 200 205

Pro Glu Thr Thr Leu Phe Leu Ile Ala Ser Lys Thr Phe Thr Thr Ala

210 215 220

Glu Thr Ile Thr Asn Ala Asn Thr Ala Lys Asn Trp Phe Leu Ser Lys 225 230 235 240

Thr Gly Asn Asp Pro Ser His Ile Ala Lys His Phe Ala Ala Leu Ser 245 250 255

Thr Asn Glu Thr Glu Val Ala Lys Phe Gly Ile Asp Thr Lys Asn Met

260 265 270

Phe Gly Phe Glu Ser Trp Val Gly Gly Arg Tyr Ser Val Trp Ser Ala 275 280 285 Ile Gly Leu Ser Val Ala Leu Tyr Ile Gly Tyr Asp Asn Phe Glu Ala

290 295 300

Phe Leu Lys Gly Ala Glu Ala Val Asp Asn His Phe Thr Gin Thr Pro 305 310 315 320

Leu Glu Asp Asn Ile Pro Leu Leu Gly Gly Leu Leu Ser Val Trp Tyr 325 330 335

Asn Asn Phe Phe Gly Ala Gin Thr His Leu Val Ala Pro Phe Asp Gin

340 345 350

Tyr Leu His Arg Phe Pro Ala Tyr Leu Gin Gin Leu Ser Met Glu Ser 355 360 365

Asn Gly Lys Ser Val Thr Arg Gly Asn Val Phe Thr Asp Tyr Ser Thr

370 375 380

Gly Ser Ile Leu Phe Gly Glu Pro Ala Thr Asn Ala Gin His Ser Phe 385 390 395 400

Phe Gin Leu Val His Gin Gly Thr Lys Leu Ile Pro Ser Asp Phe Ile 405 410 415

Leu Ala Ala Gin Ser His Asn Pro Ile Glu Asn Lys Leu His Gin Lys 420 425 430

Met Leu Ala Ser Asn Phe Phe Ala Gin Ala Glu Ala Leu Met Val Gly

435 440 445

Lys Asp Glu Glu Gin Val Lys Ala Glu Gly Ala Thr Gly Gly Leu Val

450 455 460

Pro His Lys Val Phe Ser Gly Asn Arg Pro Thr Thr Ser Ile Leu Ala 465 470 475 480

Gin Lys Ile Thr Pro Ala Thr Leu Gly Ala Leu Ile Ala Tyr Tyr Glu

485 490 495

His Val Thr Phe Thr Glu Gly Ala Ile Trp Asn Ile Asn Ser Phe Asp 500 505 510

Gin Trp Gly Val Glu Leu Gly Lys Val Leu Ala Lys Val Ile Gly Lys 515 520 525

Glu Leu Asp Asn Ser Ser Thr Ile Ser Thr His Asp Ala Ser Thr Asn

530 535 540

Gly Leu Ile Asn Gin Phe Lys Glu Trp Met 545 550

<210> 55

<211> 759

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . (759)

<223> S . cerevisiae essential gene NEP1 EMG1 YLR186W SGD1D : S000004176

<400> 55 atg gtc gaa gat tcc aga gtt aga gac gcc ctc aaa ggt ggt gat cag 48

Met Val Glu Asp Ser Arg Val Arg Asp Ala Leu Lys Gly Gly Asp Gin

1 5 10 15 aag gca tta ccg gcc tct ttg gtt cct caa gca cct cct gtc ttg aca 96

Lys Ala Leu Pro Ala Ser Leu Val Pro Gin Ala Pro Pro Val Leu Thr

20 25 30 tca aag gat aag att act aag egg atg att gtg gta tta gcg atg gca 144

Ser Lys Asp Lys Ile Thr Lys Arg Met Ile Val Val Leu Ala Met Ala

35 40 45 tcc ctc gag aca cac aag ata tcg tcc aac ggg cct ggt ggt gac aaa 192

Ser Leu Glu Thr His Lys Ile Ser Ser Asn Gly Pro Gly Gly Asp Lys

50 55 60 tat gtc ctt ttg aac tgt gac gac cat caa ggt tta tta aaa aaa atg 240

Tyr Val Leu Leu Asn Cys Asp Asp His Gin Gly Leu Leu Lys Lys Met

65 70 75 80 ggt aga gac att agt gaa gca aga cct gat att acc cac caa tgt ctt 288

Gly Arg Asp Ile Ser Glu Ala Arg Pro Asp Ile Thr His Gin Cys Leu

85 90 95 ttg acg ttg cta gat tct cca atc aac aaa gcc gga aag ctg cag gtc 336

Leu Thr Leu Leu Asp Ser Pro Ile Asn Lys Ala Gly Lys Leu Gin Val

100 105 110 tat att caa aca agt cga gga att ctg atc gag gtt aac ccc act gtt 384

Tyr Ile Gin Thr Ser Arg Gly Ile Leu Ile Glu Val Asn Pro Thr Val

115 120 125 cgt ata cca aga act ttc aaa aga ttt tca ggt tta atg gtt cag tta 432

Arg Ile Pro Arg Thr Phe Lys Arg Phe Ser Gly Leu Met Val Gin Leu

130 135 140 cta cat aag ctt tct atc aga tcg gta aat tct gaa gaa aag tta ctt 480

Leu His Lys Leu Ser Ile Arg Ser Val Asn Ser Glu Glu Lys Leu Leu

145 150 155 160 aaa gtc att aag aac cca att acc gat cac cta cct act aag tgc cgt 528

Lys Val Ile Lys Asn Pro Ile Thr Asp His Leu Pro Thr Lys Cys Arg

165 170 175 aag gtg aca tta tcc ttt gac gca cca gtt atc cgc gtt caa gat tac 576

Lys Val Thr Leu Ser Phe Asp Ala Pro Val Ile Arg Val Gin Asp Tyr

180 185 190 atc gaa aaa cta gac gat gat gaa agt ata tgt gtc ttt gtt ggt gcc 624 Ile Glu Lys Leu Asp Asp Asp Glu Ser Ile Cys Val Phe Val Gly Ala

195 200 205 atg gca aga ggt aaa gat aac ttt gcg gat gaa tac gtc gac gaa aaa 672

Met Ala Arg Gly Lys Asp Asn Phe Ala Asp Glu Tyr Val Asp Glu Lys

210 215 220 gtc ggc ttg tcc aat tac cca ttg tct gcc tca gtt gca tgt tct aaa 720 Val Gly Leu Ser Asn Tyr Pro Leu Ser Ala Ser Val Ala Cys Ser Lys

225 230 235 240 ttt tgc cat ggc gct gaa gat gct tgg aat att tta tag 759

Phe Cys His Gly Ala Glu Asp Ala Trp Asn Ile Leu

245 250

<210> 56

<211> 252

<212> PRT

<213> S . cerevisiae

<400> 56

Met Val Glu Asp Ser Arg Val Arg Asp Ala Leu Lys Gly Gly Asp Gin 1 5 10 15 Lys Ala Leu Pro Ala Ser Leu Val Pro Gin Ala Pro Pro Val Leu Thr 20 25 30

Ser Lys Asp Lys Ile Thr Lys Arg Met Ile Val Val Leu Ala Met Ala 35 40 45

Ser Leu Glu Thr His Lys Ile Ser Ser Asn Gly Pro Gly Gly Asp Lys 50 55 60

Tyr Val Leu Leu Asn Cys Asp Asp His Gin Gly Leu Leu Lys Lys Met 65 70 75 80

Gly Arg Asp Ile Ser Glu Ala Arg Pro Asp Ile Thr His Gin Cys Leu 85 90 95

Leu Thr Leu Leu Asp Ser Pro Ile Asn Lys Ala Gly Lys Leu Gin Val

100 105 110

Tyr Ile Gin Thr Ser Arg Gly Ile Leu Ile Glu Val Asn Pro Thr Val 115 120 125

Arg Ile Pro Arg Thr Phe Lys Arg Phe Ser Gly Leu Met Val Gin Leu

130 135 140

Leu His Lys Leu Ser Ile Arg Ser Val Asn Ser Glu Glu Lys Leu Leu 145 150 155 160

Lys Val Ile Lys Asn Pro Ile Thr Asp His Leu Pro Thr Lys Cys Arg 165 170 175

Lys Val Thr Leu Ser Phe Asp Ala Pro Val Ile Arg Val Gin Asp Tyr

180 185 190 Ile Glu Lys Leu Asp Asp Asp Glu Ser Ile Cys Val Phe Val Gly Ala

195 200 205

Met Ala Arg Gly Lys Asp Asn Phe Ala Asp Glu Tyr Val Asp Glu Lys

210 215 220 Val Gly Leu Ser Asn Tyr Pro Leu Ser Ala Ser Val Ala Cys Ser Lys 225 230 235 240

Phe Cys His Gly Ala Glu Asp Ala Trp Asn Ile Leu 245 250

<210> 57 <211> 804 <212> DNA <213> S . cerevisiea

<220>

<221> CDS

<222> (1) . . (804 )

<223> S . cerevisea essential gene URA3

<400> 57 atg tcg aaa gct aca tat aag gaa cgt gct gct act cat cct agt cct 48

Met Ser Lys Ala Thr Tyr Lys Glu Arg Ala Ala Thr His Pro Ser Pro

1 5 10 15 gtt gct gcc aag cta ttt aat atc atg cac gaa aag caa aca aac ttg 96 Val Ala Ala Lys Leu Phe Asn Ile Met His Glu Lys Gin Thr Asn Leu

20 25 30 tgt gct tca ttg gat gtt cgt acc acc aag gaa tta ctg gag tta gtt 144

Cys Ala Ser Leu Asp Val Arg Thr Thr Lys Glu Leu Leu Glu Leu Val

35 40 45 gaa gca tta ggt ccc aaa att tgt tta cta aaa aca cat gtg gat atc 192

Glu Ala Leu Gly Pro Lys Ile Cys Leu Leu Lys Thr His Val Asp Ile

50 55 60 ttg act gat ttt tcc atg gag ggc aca gtt aag ccg cta aag gca tta 240

Leu Thr Asp Phe Ser Met Glu Gly Thr Val Lys Pro Leu Lys Ala Leu

65 70 75 80 tcc gcc aag tac aat ttt tta ctc ttc gaa gac aga aaa ttt gct gac 288

Ser Ala Lys Tyr Asn Phe Leu Leu Phe Glu Asp Arg Lys Phe Ala Asp

85 90 95 att ggt aat aca gtc aaa ttg cag tac tct gcg ggt gta tac aga ata 336 Ile Gly Asn Thr Val Lys Leu Gin Tyr Ser Ala Gly Val Tyr Arg Ile

100 105 110 gca gaa tgg gca gac att acg aat gca cac ggt gtg gtg ggc cca ggt 384

Ala Glu Trp Ala Asp Ile Thr Asn Ala His Gly Val Val Gly Pro Gly

115 120 125 att gtt agc ggt ttg aag cag gcg gca gaa gaa gta aca aag gaa cct 432 Ile Val Ser Gly Leu Lys Gin Ala Ala Glu Glu Val Thr Lys Glu Pro

130 135 140 aga ggc ctt ttg atg tta gca gaa ttg tca tgc aag ggc tcc cta tct 480

Arg Gly Leu Leu Met Leu Ala Glu Leu Ser Cys Lys Gly Ser Leu Ser

145 150 155 160 act gga gaa tat act aag ggt act gtt gac att gcg aag agc gac aaa 528

Thr Gly Glu Tyr Thr Lys Gly Thr Val Asp Ile Ala Lys Ser Asp Lys

165 170 175 gat ttt gtt atc ggc ttt att gct caa aga gac atg ggt gga aga gat 576

Asp Phe Val Ile Gly Phe Ile Ala Gin Arg Asp Met Gly Gly Arg Asp

180 185 190 gaa ggt tac gat tgg ttg att atg aca ccc ggt gtg ggt tta gat gac 624

Glu Gly Tyr Asp Trp Leu Ile Met Thr Pro Gly Val Gly Leu Asp Asp

195 200 205 aag gga gac gca ttg ggt caa cag tat aga acc gtg gat gat gtg gtc 672 Lys Gly Asp Ala Leu Gly Gin Gin Tyr Arg Thr Val Asp Asp Val Val 210 215 220 tct aca gga tct gac att att att gtt gga aga gga cta ttt gca aag 720 Ser Thr Gly Ser Asp Ile Ile Ile Val Gly Arg Gly Leu Phe Ala Lys 225 230 235 240 gga agg gat gct aag gta gag ggt gaa cgt tac aga aaa gca ggc tgg 768 Gly Arg Asp Ala Lys Val Glu Gly Glu Arg Tyr Arg Lys Ala Gly Trp 245 250 255 gaa gca tat ttg aga aga tgc ggc cag caa aac taa 804 Glu Ala Tyr Leu Arg Arg Cys Gly Gin Gin Asn

260 265

<210> 58

<211> 267

<212> PRT

<213> S . cerevisiea

<400> 58

Met Ser Lys Ala Thr Tyr Lys Glu Arg Ala Ala Thr His Pro Ser Pro

1 5 10 15 Val Ala Ala Lys Leu Phe Asn Ile Met His Glu Lys Gin Thr Asn Leu 20 25 30

Cys Ala Ser Leu Asp Val Arg Thr Thr Lys Glu Leu Leu Glu Leu Val 35 40 45

Glu Ala Leu Gly Pro Lys Ile Cys Leu Leu Lys Thr His Val Asp Ile

50 55 60

Leu Thr Asp Phe Ser Met Glu Gly Thr Val Lys Pro Leu Lys Ala Leu 65 70 75 80

Ser Ala Lys Tyr Asn Phe Leu Leu Phe Glu Asp Arg Lys Phe Ala Asp 85 90 95 Ile Gly Asn Thr Val Lys Leu Gin Tyr Ser Ala Gly Val Tyr Arg Ile 100 105 110

Ala Glu Trp Ala Asp Ile Thr Asn Ala His Gly Val Val Gly Pro Gly

115 120 125 Ile Val Ser Gly Leu Lys Gin Ala Ala Glu Glu Val Thr Lys Glu Pro

130 135 140

Arg Gly Leu Leu Met Leu Ala Glu Leu Ser Cys Lys Gly Ser Leu Ser 145 150 155 160 Thr Gly Glu Tyr Thr Lys Gly Thr Val Asp Ile Ala Lys Ser Asp Lys

165 170 175

Asp Phe Val Ile Gly Phe Ile Ala Gin Arg Asp Met Gly Gly Arg Asp 180 185 190

Glu Gly Tyr Asp Trp Leu Ile Met Thr Pro Gly Val Gly Leu Asp Asp 195 200 205

Lys Gly Asp Ala Leu Gly Gin Gin Tyr Arg Thr Val Asp Asp Val Val

210 215 220

Ser Thr Gly Ser Asp Ile Ile Ile Val Gly Arg Gly Leu Phe Ala Lys 225 230 235 240

Gly Arg Asp Ala Lys Val Glu Gly Glu Arg Tyr Arg Lys Ala Gly Trp

245 250 255

Glu Ala Tyr Leu Arg Arg Cys Gly Gin Gin Asn 260 265

<210> 59

<211> 1095

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . (1095)

<223> S . ceverisiae essential gene LEU2

<400> 59 atg tct gcc cct aag aag atc gtc gtt ttg cca ggt gac cac gtt ggt 48 Met Ser Ala Pro Lys Lys Ile Val Val Leu Pro Gly Asp His Val Gly 1 5 10 15 caa gaa atc aca gcc gaa gcc att aag gtt ctt aaa gct att tct gat 96

Gin Glu Ile Thr Ala Glu Ala Ile Lys Val Leu Lys Ala Ile Ser Asp

20 25 30 gtt cgt tec aat gtc aag ttc gat ttc gaa aat cat tta att ggt ggt 144 Val Arg Ser Asn Val Lys Phe Asp Phe Glu Asn His Leu Ile Gly Gly

35 40 45 gct gct atc gat gct aca ggt gtc cca ctt cca gat gag gcg ctg gaa 192

Ala Ala Ile Asp Ala Thr Gly Val Pro Leu Pro Asp Glu Ala Leu Glu

50 55 60 gcc tcc aag aag gtt gat gcc gtt ttg tta ggt gct gtg gct ggt cct 240

Ala Ser Lys Lys Val Asp Ala Val Leu Leu Gly Ala Val Ala Gly Pro

65 70 75 80 aaa tgg ggt acc ggt agt gtt aga cct gaa caa ggt tta cta aaa atc 288

Lys Trp Gly Thr Gly Ser Val Arg Pro Glu Gin Gly Leu Leu Lys Ile

85 90 95 cgt aaa gaa ctt caa ttg tac gcc aac tta aga cca tgt aac ttt gca 336

Arg Lys Glu Leu Gin Leu Tyr Ala Asn Leu Arg Pro Cys Asn Phe Ala

100 105 110 tcc gac tct ctt tta gac tta tct cca atc aag cca caa ttt gct aaa 384

Ser Asp Ser Leu Leu Asp Leu Ser Pro Ile Lys Pro Gin Phe Ala Lys

115 120 125 ggt act gac ttc gtt gtt gtc aga gaa tta gtg gga ggt att tac ttt 432

Gly Thr Asp Phe Val Val Val Arg Glu Leu Val Gly Gly Ile Tyr Phe

130 135 140 ggt aag aga aag gaa gac gat ggt gat ggt gtc gct tgg gat agt gaa 480

Gly Lys Arg Lys Glu Asp Asp Gly Asp Gly Val Ala Trp Asp Ser Glu

145 150 155 160 caa tac acc gtt cca gaa gtg caa aga atc aca aga atg gcc gct ttc 528

Gin Tyr Thr Val Pro Glu Val Gin Arg Ile Thr Arg Met Ala Ala Phe

165 170 175 atg gcc cta caa cat gag cca cca ttg cct att tgg tcc ttg gat aaa 576

Met Ala Leu Gin His Glu Pro Pro Leu Pro Ile Trp Ser Leu Asp Lys

180 185 190 gct aat ctt ttg gcc tct tca aga tta tgg aga aaa act gtg gag gaa 624

Ala Asn Leu Leu Ala Ser Ser Arg Leu Trp Arg Lys Thr Val Glu Glu

195 200 205 acc atc aag aac gaa ttc cct aca ttg aag gtt caa cat caa ttg att 672

Thr Ile Lys Asn Glu Phe Pro Thr Leu Lys Val Gin His Gin Leu Ile

210 215 220 gat tct gcc gcc atg atc cta gtt aag aac cca acc cac cta aat ggt 720

Asp Ser Ala Ala Met Ile Leu Val Lys Asn Pro Thr His Leu Asn Gly

225 230 235 240 att ata atc acc agc aac atg ttt ggt gat atc atc tcc gat gaa gcc 768 Ile Ile Ile Thr Ser Asn Met Phe Gly Asp Ile Ile Ser Asp Glu Ala

245 250 255 tcc gtt atc cca ggt tcc ttg ggt ttg ttg cca tct gcg tcc ttg gcc 816

Ser Val Ile Pro Gly Ser Leu Gly Leu Leu Pro Ser Ala Ser Leu Ala

260 265 270 tct ttg cca gac aag aac acc gca ttt ggt ttg tac gaa cca tgc cac 864

Ser Leu Pro Asp Lys Asn Thr Ala Phe Gly Leu Tyr Glu Pro Cys His

275 280 285 ggt tct gct cca gat ttg cca aag aat aag gtt gac cct atc gcc act 912

Gly Ser Ala Pro Asp Leu Pro Lys Asn Lys Val Asp Pro Ile Ala Thr

290 295 300 atc ttg tct gct gca atg atg ttg aaa ttg tca ttg aac ttg cct gaa 960 Ile Leu Ser Ala Ala Met Met Leu Lys Leu Ser Leu Asn Leu Pro Glu

305 310 315 320 gaa ggt aag gcc att gaa gat gca gtt aaa aag gtt ttg gat gca ggt 1008

Glu Gly Lys Ala Ile Glu Asp Ala Val Lys Lys Val Leu Asp Ala Gly

325 330 335 atc aga act ggt gat tta ggt ggt tcc aac agt acc acc gaa gtc ggt 1056 Ile Arg Thr Gly Asp Leu Gly Gly Ser Asn Ser Thr Thr Glu Val Gly 340 345 350 gat gct gtc gcc gaa gaa gtt aag aaa atc ctt gct taa 1095 Asp Ala Val Ala Glu Glu Val Lys Lys Ile Leu Ala 355 360

<210> 60

<211> 364

<212> PRT

<213> S . cerevisiae

<400> 60

Met Ser Ala Pro Lys Lys Ile Val Val Leu Pro Gly Asp His Val Gly

1 5 10 15

Gin Glu Ile Thr Ala Glu Ala Ile Lys Val Leu Lys Ala Ile Ser Asp

20 25 30 Val Arg Ser Asn Val Lys Phe Asp Phe Glu Asn His Leu Ile Gly Gly 35 40 45

Ala Ala Ile Asp Ala Thr Gly Val Pro Leu Pro Asp Glu Ala Leu Glu 50 55 60

Ala Ser Lys Lys Val Asp Ala Val Leu Leu Gly Ala Val Ala Gly Pro 65 70 75 80

Lys Trp Gly Thr Gly Ser Val Arg Pro Glu Gin Gly Leu Leu Lys Ile 85 90 95

Arg Lys Glu Leu Gin Leu Tyr Ala Asn Leu Arg Pro Cys Asn Phe Ala 100 105 110

Ser Asp Ser Leu Leu Asp Leu Ser Pro Ile Lys Pro Gin Phe Ala Lys 115 120 125

Gly Thr Asp Phe Val Val Val Arg Glu Leu Val Gly Gly Ile Tyr Phe 130 135 140

Gly Lys Arg Lys Glu Asp Asp Gly Asp Gly Val Ala Trp Asp Ser Glu 145 150 155 160

Gin Tyr Thr Val Pro Glu Val Gin Arg Ile Thr Arg Met Ala Ala Phe

165 170 175

Met Ala Leu Gin His Glu Pro Pro Leu Pro Ile Trp Ser Leu Asp Lys 180 185 190

Ala Asn Leu Leu Ala Ser Ser Arg Leu Trp Arg Lys Thr Val Glu Glu 195 200 205

Thr Ile Lys Asn Glu Phe Pro Thr Leu Lys Val Gin His Gin Leu Ile

210 215 220

Asp Ser Ala Ala Met Ile Leu Val Lys Asn Pro Thr His Leu Asn Gly 225 230 235 240 Ile Ile Ile Thr Ser Asn Met Phe Gly Asp Ile Ile Ser Asp Glu Ala

245 250 255

Ser Val Ile Pro Gly Ser Leu Gly Leu Leu Pro Ser Ala Ser Leu Ala 260 265 270

Ser Leu Pro Asp Lys Asn Thr Ala Phe Gly Leu Tyr Glu Pro Cys His 275 280 285

Gly Ser Ala Pro Asp Leu Pro Lys Asn Lys Val Asp Pro Ile Ala Thr

290 295 300 Ile Leu Ser Ala Ala Met Met Leu Lys Leu Ser Leu Asn Leu Pro Glu

305 310 315 320

Glu Gly Lys Ala Ile Glu Asp Ala Val Lys Lys Val Leu Asp Ala Gly

325 330 335 Ile Arg Thr Gly Asp Leu Gly Gly Ser Asn Ser Thr Thr Glu Val Gly

340 345 350

Asp Ala Val Ala Glu Glu Val Lys Lys Ile Leu Ala 355 360

<210> 61

<211> 675

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . ( 675)

<223> S . cerevisiae essential gene TRP1

<400> 61 atg tct gtt att aat ttc aca ggt agt tct ggt cca ttg gtg aaa gtt 48

Met Ser Val Ile Asn Phe Thr Gly Ser Ser Gly Pro Leu Val Lys Val

1 5 10 15 tgc ggc ttg cag agc aca gag gcc gca gaa tgt gct cta gat tec gat 96

Cys Gly Leu Gin Ser Thr Glu Ala Ala Glu Cys Ala Leu Asp Ser Asp

20 25 30 gct gac ttg ctg ggt att ata tgt gtg ccc aat aga aag aga aca att 144 Ala Asp Leu Leu Gly Ile Ile Cys Val Pro Asn Arg Lys Arg Thr Ile 35 40 45 gac ccg gtt att gca agg aaa att tca agt ctt gta aaa gca tat aaa 192

Asp Pro Val Ile Ala Arg Lys Ile Ser Ser Leu Val Lys Ala Tyr Lys

50 55 60 aat agt tca ggc act ccg aaa tac ttg gtt ggc gtg ttt cgt aat caa 240

Asn Ser Ser Gly Thr Pro Lys Tyr Leu Val Gly Val Phe Arg Asn Gin

65 70 75 80 cct aag gag gat gtt ttg gct ctg gtc aat gat tac ggc att gat atc 288

Pro Lys Glu Asp Val Leu Ala Leu Val Asn Asp Tyr Gly Ile Asp Ile

85 90 95 gtc caa ctg cat gga gat gag tcg tgg caa gaa tac caa gag ttc ctc 336 Val Gin Leu His Gly Asp Glu Ser Trp Gin Glu Tyr Gin Glu Phe Leu

100 105 110 ggt ttg cca gtt att aaa aga ctc gta ttt cca aaa gac tgc aac ata 384

Gly Leu Pro Val Ile Lys Arg Leu Val Phe Pro Lys Asp Cys Asn Ile

115 120 125 cta ctc agt gca gct tca cag aaa cct cat tcg ttt att ccc ttg ttt 432

Leu Leu Ser Ala Ala Ser Gin Lys Pro His Ser Phe Ile Pro Leu Phe

130 135 140 gat tca gaa gca ggt ggg aca ggt gaa ctt ttg gat tgg aac tcg att 480

Asp Ser Glu Ala Gly Gly Thr Gly Glu Leu Leu Asp Trp Asn Ser Ile

145 150 155 160 tct gac tgg gtt gga agg caa gag agc ccc gaa agc tta cat ttt atg 528

Ser Asp Trp Val Gly Arg Gin Glu Ser Pro Glu Ser Leu His Phe Met

165 170 175 tta gct ggt gga ctg acg cca gaa aat gtt ggt gat gcg ctt aga tta 576

Leu Ala Gly Gly Leu Thr Pro Glu Asn Val Gly Asp Ala Leu Arg Leu

180 185 190 aat ggc gtt att ggt gtt gat gta agc gga ggt gtg gag aca aat ggt 624

Asn Gly Val Ile Gly Val Asp Val Ser Gly Gly Val Glu Thr Asn Gly

195 200 205 gta aaa gac tct aac aaa ata gca aat ttc gtc aaa aat gct aag aaa 672 Val Lys Asp Ser Asn Lys Ile Ala Asn Phe Val Lys Asn Ala Lys Lys

210 215 220 tag 675

<210> 62

<211> 224

<212> PRT

<213> S . cerevisiae

<400> 62

Met Ser Val Ile Asn Phe Thr Gly Ser Ser Gly Pro Leu Val Lys Val

1 5 10 15

Cys Gly Leu Gin Ser Thr Glu Ala Ala Glu Cys Ala Leu Asp Ser Asp 20 25 30

Ala Asp Leu Leu Gly Ile Ile Cys Val Pro Asn Arg Lys Arg Thr Ile 35 40 45

Asp Pro Val Ile Ala Arg Lys Ile Ser Ser Leu Val Lys Ala Tyr Lys 50 55 60

Asn Ser Ser Gly Thr Pro Lys Tyr Leu Val Gly Val Phe Arg Asn Gin 65 70 75 80

Pro Lys Glu Asp Val Leu Ala Leu Val Asn Asp Tyr Gly Ile Asp Ile 85 90 95 Val Gin Leu His Gly Asp Glu Ser Trp Gin Glu Tyr Gin Glu Phe Leu 100 105 110

Gly Leu Pro Val Ile Lys Arg Leu Val Phe Pro Lys Asp Cys Asn Ile 115 120 125

Leu Leu Ser Ala Ala Ser Gin Lys Pro His Ser Phe Ile Pro Leu Phe 130 135 140

Asp Ser Glu Ala Gly Gly Thr Gly Glu Leu Leu Asp Trp Asn Ser Ile 145 150 155 160

Ser Asp Trp Val Gly Arg Gin Glu Ser Pro Glu Ser Leu His Phe Met

165 170 175

Leu Ala Gly Gly Leu Thr Pro Glu Asn Val Gly Asp Ala Leu Arg Leu 180 185 190

Asn Gly Val Ile Gly Val Asp Val Ser Gly Gly Val Glu Thr Asn Gly 195 200 205 Val Lys Asp Ser Asn Lys Ile Ala Asn Phe Val Lys Asn Ala Lys Lys 210 215 220

<210> 63

<211> 657

<212> DNA

<213> S . cerevisiae

<220>

<221> CDS

<222> (1) . . ( 657 )

<223> S . cerevisiae essential gene HIS3

<400> 63 atg aca gag cag aaa gcc cta gta aag cgt att aca aat gaa acc aag 48 Met Thr Glu Gin Lys Ala Leu Val Lys Arg Ile Thr Asn Glu Thr Lys 1 5 10 15 att cag att gcg atc tct tta aag ggt ggt ccc cta gcg ata gag cac 96 Ile Gin Ile Ala Ile Ser Leu Lys Gly Gly Pro Leu Ala Ile Glu His

20 25 30 tcg atc ttc cca gaa aaa gag gca gaa gca gta gca gaa cag gcc aca 144

Ser Ile Phe Pro Glu Lys Glu Ala Glu Ala Val Ala Glu Gin Ala Thr

35 40 45 caa tcg caa gtg att aac gtc cac aca ggt ata ggg ttt ctg gac cat 192

Gin Ser Gin Val Ile Asn Val His Thr Gly Ile Gly Phe Leu Asp His

50 55 60 atg ata cat gct ctg gcc aag cat tcc ggc tgg tcg cta atc gtt gag 240

Met Ile His Ala Leu Ala Lys His Ser Gly Trp Ser Leu Ile Val Glu

65 70 75 80 tgc att ggt gac tta cac ata gac gac cat cac acc act gaa gac tgc 288

Cys Ile Gly Asp Leu His Ile Asp Asp His His Thr Thr Glu Asp Cys

85 90 95 ggg att gct ctc ggt caa gct ttt aaa gag gcc cta ctg gcg cgt gga 336

Gly Ile Ala Leu Gly Gin Ala Phe Lys Glu Ala Leu Leu Ala Arg Gly

100 105 110 gta aaa agg ttt gga tca gga ttt gcg cct ttg gat gag gca ctt tcc 384 Val Lys Arg Phe Gly Ser Gly Phe Ala Pro Leu Asp Glu Ala Leu Ser

115 120 125 aga gcg gtg gta gat ctt tcg aac agg ccg tac gca gtt gtc gaa ctt 432

Arg Ala Val Val Asp Leu Ser Asn Arg Pro Tyr Ala Val Val Glu Leu

130 135 140 ggt ttg caa agg gag aaa gta gga gat ctc tct tgc gag atg atc ccg 480

Gly Leu Gin Arg Glu Lys Val Gly Asp Leu Ser Cys Glu Met Ile Pro

145 150 155 160 cat ttt ctt gaa agc ttt gca gag gct agc aga att acc ctc cac gtt 528

His Phe Leu Glu Ser Phe Ala Glu Ala Ser Arg Ile Thr Leu His Val

165 170 175 gat tgt ctg cga ggc aag aat gat cat cac cgt agt gag agt gcg ttc 576

Asp Cys Leu Arg Gly Lys Asn Asp His His Arg Ser Glu Ser Ala Phe

180 185 190 aag gct ctt gcg gtt gcc ata aga gaa gcc acc tcg ccc aat ggt acc 624

Lys Ala Leu Ala Val Ala Ile Arg Glu Ala Thr Ser Pro Asn Gly Thr

195 200 205 aac gat gtt ccc tcc acc aaa ggt gtt ctt atg 657 Asn Asp Val Pro Ser Thr Lys Gly Val Leu Met 210 215

<210> 64

<211> 219

<212> PRT

<213> S . cerevisiae

<400> 64 Met Thr Glu Gin Lys Ala Leu Val Lys Arg Ile Thr Asn Glu Thr Lys 1 5 10 15 Ile Gin Ile Ala Ile Ser Leu Lys Gly Gly Pro Leu Ala Ile Glu His 20 25 30

Ser Ile Phe Pro Glu Lys Glu Ala Glu Ala Val Ala Glu Gin Ala Thr 35 40 45

Gin Ser Gin Val Ile Asn Val His Thr Gly Ile Gly Phe Leu Asp His 50 55 60

Met Ile His Ala Leu Ala Lys His Ser Gly Trp Ser Leu Ile Val Glu 65 70 75 80

Cys Ile Gly Asp Leu His Ile Asp Asp His His Thr Thr Glu Asp Cys 85 90 95

Gly Ile Ala Leu Gly Gin Ala Phe Lys Glu Ala Leu Leu Ala Arg Gly 100 105 110 Val Lys Arg Phe Gly Ser Gly Phe Ala Pro Leu Asp Glu Ala Leu Ser 115 120 125

Arg Ala Val Val Asp Leu Ser Asn Arg Pro Tyr Ala Val Val Glu Leu 130 135 140

Gly Leu Gin Arg Glu Lys Val Gly Asp Leu Ser Cys Glu Met Ile Pro 145 150 155 160

His Phe Leu Glu Ser Phe Ala Glu Ala Ser Arg Ile Thr Leu His Val

165 170 175

Asp Cys Leu Arg Gly Lys Asn Asp His His Arg Ser Glu Ser Ala Phe 180 185 190

Lys Ala Leu Ala Val Ala Ile Arg Glu Ala Thr Ser Pro Asn Gly Thr 195 200 205

Asn Asp Val Pro Ser Thr Lys Gly Val Leu Met 210 215

<210> 65

<211> 290

<212> DNA

<213> E . coli <220>

<221> promoter

<222> (1) • • (290)

<223> burden sensing promoter p (htpG)

<400> 65 gccaccaacc ccacggttga aggtgaagct accgctaact acattgccga gctttgcgcg 60 caatatgacg tggaagccag ccgaatcgct catggcgtac cggttggcgg cgaactggaa 120 atggtcgatg gcaccacgct gtcacactcc cttgccgggc gtcataagat tcgtttttaa 180 gcaaacgaga gcaggatcac ctgctctcgc ttgaaattat tctcccttgt ccccatctct 240 ccctcatcct gtttttaacc ttaaaatggc attattgagg tagacctaca 290

<210> 66

<211> 257

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (257 )

<223> burden sensing promoter p (ibpA)

<400> 66 atcccttgct gaaaataaca tcatcattac gtcgcactgt ggcggctatc gcactttaac 60 gtttcgtgct gccccctcag tctatgcaat agaccataaa ctgcaaaaaa aagtccgctg 120 ataaggcttg aaaagttcat ttccagaccc atttttacat cgtagccgat gaggacgcgc 180 ctgatgggtg ttctggctac ctgacctgtc cattgtggaa ggtcttacat tctcgctgat 240 ttcaggagct attgatt 257

<210> 67

<211> 298

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (298)

<223> burden sensing promoter p (clpB)

<400> 67 gataagtatc tggcggatat ttatctgctt gcccggcagc gtctggcgag cgttggtgtt 60 gaacaaattt tcggcggcga ccgttgtaca tatacggaaa atgagacttt cttctcttat 120 cgtcgcgaca agaccaccgg gcgtatggca agtttcattt ggctgatata acctaaagaa 180 tcaagacgat ccggtacgcg tgattttctt ttcacattaa tctggtcaat aaccttgaat 240 aattgaggga tgacctcatt taatctccag tagcaacttt gatccgttat gggaggag 298 <210> 68

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (yccV)

<400> 68 ggttataaag acatcaacat gctggcgatt cagctgctga atgaaggcgg tattctcctg 60 actttctcct gttccggtct gatgaccagc gatttatttc agaaaatcat cgcggatgcc 120 gcaattgatg ccggtcgtga tgtacaattt atagagcagt tccgtcaggc agccgatcat 180 ccggtgatcg ctacctatcc ggaagggcta tatctgaaag ggtttgcctg tcgcgtcatg 240 taacttgaaa tgtggaattt tacccttata tcaaaggtaa agatttcccg ggaggtgact 300

<210> 69

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (grpE)

<400> 69 tctccgcgag cgtgccagtt ttcacattct tcagttgcag ttcgtgagcg atttgttgct 60 caacgatgac ctcgtaacct tttgtgcaca gccagcggta gagcatttca tgtgttgtca 120 gtgcagtggg gtgccgtggg tgtcccacaa tgccaataca cttgaaatga ttattcattt 180 ttccgaggtc cttgttgcga agattgatga caatgtgagt gcttcccttg aaaccctgaa 240 actgatcccc ataataagcg aagttagcga gatgaatgcg aaaaaaacgc ggagaaattc 300

<210> 70

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (yc jX)

<400> 70 ttggctgata ctgtaattct tcaatggccc gtaacacttt ttcgcgcgct tccggggaga 60 tattagtttg cttattcagc acgcgtgata cggtggattt tgatacgcct gcaaccctgg 120 caatatcata aatagtaggg gacataggtc aggaactccg tccgaacgtg aatgctgcac 180 atcttatgga gttcgccggt cgatgacaac acgggcagtt gataatcaat ggcctggccc 240 ccacattcat atccttacga atgatttttt ttctggtctt cagagcagca caggacagca 300

<210> 71

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (ldhA)

<400> 71 gggaacccac agcccgagcg tcatcagcag cgtcaacggc acaagaataa tcagtaataa 60 cagcgcgaga acggctttat atttacccag catgggtagt taatatcctg atttagcgaa 120 aaattaagca ttcaatacgg gtattgtggc atgtttaacc gttcagttga aggttgcgcc 180 tacactaagc atagttgttg atgaattttt caatatcgcc atagctttca attatatttg 240 aaattttgta aaatattttt agtagcttaa atgtgattca acatcactgg agaaagtctt 300

<210> 72

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (mutM)

<400> 72 ggcgaaaagt actaagtact tagaggaaat aaatcatggc taaaggtatt cgtgagaaaa 60 tcaagctggt ttcttctgct ggtactggtc acttctatac cactacgaag aacaaacgta 120 ctaagccgga aaaactggaa ctgaaaaaat tcgatccagt tgttcgccag cacgtgatct 180 acaaagaagc gaaaatcaaa taattctcgc tttgatgtaa caaaaaacct cgctccggcg 240 gggttttttg ttatctgctt gcccccatat tgactgcatc tgttcattcc tggagatgct 300

<210> 73

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (ybbN) <400> 73 cccattcgta ctcgcttcac cgacaacgtc aaccagacgc aaagtgataa accagtcgaa 60 aatcccggca tcgccgcccg ctttacgttg ggaccggaag cggtggtgga caaagtacgc 120 catgctttta ttagcgagaa gccgaagatg cgctatccgg taacgctggt gacctgggca 180 gtaatggtgc ttaagcgcct gctgccgggg cgcgtgatgg acaaaatatt gcaggggtga 240 gttgaagcgc gcgcttaagc ccccatgtca aagaaaatat cgacaacaga gagcgactcc 300

<210> 74

<211> 298

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (298)

<223> burden sensing promoter p (prlC)

<400> 74 agttcttgtt tgagtccact catgggttga tgtccgattg cgcccaaatt ttgggcaact 60 gcgtagattt tcgatggtag cacaatcaga ttcgcttatg acggtgatga agaaattgcg 120 atgaaatgtg aggtgaatca gggttttcac ccgattttgt gctgatcaga attttttttc 180 tttttccccc ttgaaggggc gaagcctcat ccccatttct ctggtcacca gccgggaaac 240 cacgtaagct ccggcgtcac ccataacaga tacggacttt ctcaaaggag agttatca 298

<210> 75

<211> 298

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (298)

<223> burden sensing promoter p (groES)

<400> 75 agttcttgtt tgagtccact catgggttga tgtccgattg cgcccaaatt ttgggcaact 60 gcgtagattt tcgatggtag cacaatcaga ttcgcttatg acggtgatga agaaattgcg 120 atgaaatgtg aggtgaatca gggttttcac ccgattttgt gctgatcaga attttttttc 180 tttttccccc ttgaaggggc gaagcctcat ccccatttct ctggtcacca gccgggaaac 240 cacgtaagct ccggcgtcac ccataacaga tacggacttt ctcaaaggag agttatca 298

<210> 76

<211> 392

<212> DNA

<213> E . coli <220>

<221> promoter

<222> (1) • • (392 )

<223> burden sensing promoter p (fxsA)

<220>

<221> misc_feature

<222> (328) . . (328)

<223> N is A, T, C or G

<400> 76 gatccccgga atattcatat gttaaacaca cagaatatat gtggtttcga atgtttttcg 60 accgacgatt atcccctgca tcgaccgaat acccgagatc atatgctgct tgaggatttc 120 taccgtaatc tggatcactt taagtgtcgg tttttacccc ttaattatta atttgtgaaa 180 tagatcaccg ctttgggatt actaccaaaa atagttgcgc aaacatcttg aaattttgct 240 aatgaccaca atataagctg aacgcgattc gcaacccatt caggtagccg ggttttaccg 300 gctgctatta caggagaaac ctacttgnaa ggtatgaaac tctttgccca gggtacttca 360 ctggacctta gccatcctca cgtaatgggg at 392

<210> 77

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (htpX)

<400> 77 agattaccag gagccggatc cttatctgga tgagacggtg aatatcgcac tcgatctggc 60 gaagcttgaa aaagccagac ccgcggaaca acccgctccc gtcaagtaat atcaatcagg 120 cacaagaaat tgtgcctgat tttttaacag cggcaagatg ccgtaaatca gatgctacaa 180 aatgtaaagt tgtgtctttc tggtgactta cgcactatcc agacttgaaa atagtcgcgt 240 aacccatacg atgtgggtat cgcatattgc gttttgttaa actgaggtaa aaagaaaatt 300

<210> 78

<211> 275

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (275)

<223> burden sensing promoter p (rrnB)

<400> 78 gcaaacacgc cgccgggtca gcggggttct cctgagaatt ccggcagaga aagcaaaaat 60 aaatgcttga ctctgtagcg ggaaagcgta ttatgcacac cccgcgccgc tgagaaaaag 120 cgaagcggca ctgctcttta acaatttatc agacaatctg tgtgggcact cgaagatacg 180 gattcttaac gtcgcaagac gaaaaatgaa taccaagtct caagagtgaa cacgtaattc 240 attacgaagt ttaattcttt gagcgtcaaa ctttt 275

<210> 79

<211> 296

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (296)

<223> burden sensing promoter p (rrnE)

<400> 79 aatgcgcctc cgttgagacg acaacgtgaa acacttcaca ggatggtcgg aacaacgaag 60 agaaaaaaat cctgaaattc agggttgact ctgaaagagg aaagcgtaat atacgccacc 120 tcgcgacagt gagctgaaag ccgcgtcgca actgctcttt aacaatttat cagacaatct 180 gtgtgggcac tcgaagatac ggattcttaa cgtcgcaaga cgaaaaatga ataccaagtc 240 tcaagagtga acacgtaatt cattacgaag tttaattctt tgagcatcaa actttt 296

<210> 80

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (cspD)

<400> 80 cggttatcgg cagaacgccc tgccgctgac aagagcttat ttgcctgacc aatctactcc 60 acatgataga taactatcat ctatcgttgg catcagcgac atctgtcaca ttcctgtcaa 120 tagcgttaac tgcttcaaat ttttgattca tacctgcccc ctgtcccctg tcaaatgctt 180 gacggctcgc cctaattctc taaattgtat ttctagagtt ggcgaggttt tgaacagccc 240 cctctctgac cccggtttat tccatcttac ttgtataaga tttgcgaagg atgtcgaagc 300

<210> 81

<211> 299

<212> DNA

<213> E . coli

<220> <221> promoter

<222> (1) • • (299)

<223> burden sensing promoter p (katE)

<400> 81 taaatccatc tttatcgcat gttctggcgg cgcgggttcc gtgcgtggga catagctaat 60 aatctggcgg ttttgctggc ggagcggttt cttcattact ggcttcacta aacgcatatt 120 aaaaatcaga aaaactgtag tttagccgat ttagcccctg tacgtcccgc tttgcgtgta 180 tttcataaca ccgtttccag aatagtctcc gaagcgggat ctggctggtg gtctatagtt 240 agagagtttt ttgaccaaaa cagcggccct ttcagtaata aattaaggag acgagttca 299

<210> 82

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (xthA)

<400> 82 caaaccatat gcatttaaag tgcatataaa gtgaatacgt ttgcgatgtg ggtgaataaa 60 aagaataaaa aacgcaatgt tatgcagaag taaaatataa ttctggaatt gtgatcattg 120 acgaaattta ctggaaatta ctgcgccatt ctgacgcagc gcgcaccaaa agcgggcatt 180 ttttgcgcca tcgttgacat cattaacaat catcgatcaa atcacttaac aacaggcggt 240 aagcaacgcg aaattctgct accatccacg cactctttat ctgaataaat ggcagcgact 300

<210> 83

<211> 301

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (301)

<223> burden sensing promoter p (uspE)

<400> 83 taatatctgg gcctgacggt agaaaccatc agccgtctgc tgggtcgctt ccagaaaagc 60 ggtatgctgg cagtcaaagg taaatacatc accatcgaaa ataacgatgc gctggcccag 120 cttgctggtc atacgcgtaa cgttgcctga tttttccgca taactcacta tccttctgtc 180 atatcattaa atttttctga tttattgatc tggcagaagg ttcatcactg tttcattcac 240 cagatatggg ttaatctttt aattacaaac tgcgttgaca gttgttgtaa ggagaccctg 300 t 301 <210> 84

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (gadB)

<400> 84 taattttata aatgcgttca aaataataat caagtactaa tagtgatatt ttaaggtctg 60 atttttacgt gataattcag gaggcacaga atgcgcataa aaataacagc ataaaacacc 120 ttaccaccac ccaagaattt catattgtat tgtttttcaa tgaaaaaata ttattcgcgt 180 aatatctcac gataaataac attaggattt tgttatttaa acacgagtcc tttgcacttg 240 cttactttat cgataaatcc tactttttta atgcgatcca atcattttaa ggagtttaaa 300

<210> 85

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (ahpC)

<400> 85 tagatcaggt gattgccctt tgtttatgag ggtgttgtaa tccatgtcgt tgttgcattt 60 gtaagggcaa cacctcagcc tgcaggcagg cactgaagat accaaagggt agttcagatt 120 acacggtcac ctggaaaggg ggccatttta ctttttatcg ccgctggcgg tgcaaagttc 180 acaaagttgt cttacgaagg ttgtaaggta aaacttatcg atttgataat ggaaacgcat 240 tagccgaatc ggcaaaaatt ggttacctta catctcatcg aaaacacgga ggaagtatag 300

<210> 86

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (katG)

<400> 86 gtgatcacaa attttaaaca gagcacaaaa tgctgcctcg aaatgagggc gggaaaataa 60 ggttatcagc cttgttttct ccctcattac ttgaaggata tgaagctaaa accctttttt 120 ataaagcatt tgtccgaatt cggacataat caaaaaagct taattaagat caatttgatc 180 tacatctctt taaccaacaa tatgtaagat ctcaactatc gcatccgtgg attaattcaa 240 ttataacttc tctctaacgc tgtgtatcgt aacggtaaca ctgtagaggg gagcacattg 300

<210> 87

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (grxA)

<400> 87 tagaatctgc acttctggcc gagaaaatgg ctcaggcagc gacagggagt cgcttaccga 60 cagcagcgcc accgccagta gcatcattcc gataaattcc agtatcaaca cgcctttagg 120 caatttaccg atcgcgcgca tacgcttccc tctgcaaagt gagccttcag tctaaaactt 180 ttcactgtat tgtgtttaac agttatagct tttagcaatt aatgcaacag gttaaaccta 240 ctttcagcga atacatttta gcgtgatcat tacaggcata aatctatgag gagagaaata 300

<210> 88

<211> 299

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (299)

<223> burden sensing promoter p (oxyS)

<400> 88 taactgatcc accagcagca ttcccgcctg ggtgaacaac actttacggc tggtccgctc 60 cagcaacatc acgcccagct catcttccag cttacgaatt tgcccgctaa gcgtcggctg 120 gctaacgtgg caggaatctg ccgcacgccg aaaatggcgg tgttcagcca atgccaccag 180 gtactcaaga tcacgaatat tcattatcca tcctccatcg ccacgatagt tcatggcgat 240 aggtagaata gcaatgaacg attatcccta tcaagcattc tgactgataa ttgctcaca 299

<210> 89

<211> 300

<212> DNA

<213> E . coli

<220>

<221> promoter

<222> (1) • • (300)

<223> burden sensing promoter p (poxB) <400> 89 taggttgtcg ctgcctgccg tgcgggtgtt tgcggctgct gtaagacaaa agtggtttcc 60 ggtgaatata cggtgagcag cacaatgacg ctgaccgacg ccgaaatcgc tgaaggttac 120 gtactggcct gctcctgcca tccgcagggg gatttggttc tcgcataatc gccttatgcc 180 cgatgatatt cctttcatcg ggctatttaa ccgttagtgc ctcctttctc tcccatccct 240 tccccctccg tcagatgaac taaacttgtt accgttatca cattcaggag atggagaacc 300

<210> 90

<211> 299

<212> DNA

<213> E . coll

<220>

<221> promoter

<222> (1) • • (299)

<223> burden sensing promoter p (trxC)

<400> 90 tagcgggttg tgggtacgtt tatcagaatc atcatcactg cggacataca tcactttgac 60 cttgccgctt ttacctttca tttcatcgtt catgcttttc tccaccagcg ctgcgcgaag 120 cgcgcagatt acccgaagtc cgcgcggttc gccatgattt cgtaccaaag cctgcgacta 180 tcatacctat tgaataaaac agattgttgt ctggaacaat gtccccgata atatgtaaca 240 tattagaaac ataccggcgt cgttgccgat aagtctcctt actcatcccg aggttagtt 299

<210> 91

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pKAR2

<400> 91 gtgggagtca atcaaatccc aatcgacgct attactggac aagtcaactt tattcgcaaa 60 acaagtcctg taatcatcac caaaagtctc aatgaatgct tcttcattaa gatactttga 120 ccattccata ggcggaaacg tgtttttatc caacactctg tcactcttgg accgtggctt 180 tggcgccctt tcattttgtc ttacataaaa gtatctcagc agcattattg cttctttcct 240 caagatcccc ggtatactct cgtaccctgc cgcactattg ttcttgggca ctaatgtaca 300 cgtatcatga tttactgaca agacagtacc gttgcctcca aatctctcgt tgccacaacc 360 gaacaccacc tttccaatgt ctaattgctt gagagcagat gcacacatta tacacggttc 420 tacagtaaca tatagagtaa tgtctttgaa cacgtcaaca actcctcggg agcccaacat 480 cgccttgatc tgatcgatcc ccatgaactc agcatgtgct actccagtta atgacttgtt 540 cgtatcgttc atgccataag ccatcacctg gccagttggc gtatgtacaa agatgcaagc 600 taccggtgtc tcatcgtggt caagagcgta tctagccaaa cggacagctg tcctcatatg 660 tttaatatgc tgcatagtgt gagtcctcta gtttttaccg cagccaccag ccgcttctcg 720 agcaaagtgt agatcccatt aggactcatc attcatctaa ttttgctatg ttagctgcaa 780 ctttctattt taatagaacc ttctggaaat ttcacccggc gcggcacccg aggaactgga 840 cagcgtgtcg aaaaagttgc ttttttatat aaaggacacg aaaagggttc tctggaagat 900 ataaatatgg ctatgtaatt ctaaagatta acgtgttact gttttacttt tttaaagtcc 960 ccaagagtag tctcaaggga aaaagcgtat caaacatacc 1000

<210> 92

<211> 1000

<212> DNA

<213> S . cerevisiea

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pPDIl

<400> 92 caggtcgtca gatgtgacgt tctcatcgtc tagcaaatca tcgggaatct tggacttcat 60 ttgctccatg gagaaagtat gatctccatg caagttaaca gaacatatac ggaaattggt 120 accacccagg tcggcggcta gtaaaacacc gcgctccgtc ccgttgggtg acccggtgac 180 gaacgccgga atcataggaa gacctttgtc cgaggccaat gtgtggcctt cctttggtgg 240 agctagacct ttttccattt gttcgatgaa gtaagcagtt aattcgtcca gcttctcggg 300 ggtaacctcg aaatcgtcgc agatctggtc cacggcctgg atgaccgctc tctcagtggc 360 tttgtgtaag tcgtcgaatg acatcttgtg tatgatagag ttgtattagt ggtggtgttg 420 tggtttacgg aagctgagtt gggcactgat gggggggttg ggggttctgt tgatagcgga 480 tatttatatg ggcgcgtttt cattagtgcc caccgtttga gcgtggtgtg acaccacgcc 540 caagataggg ggccccctga aaggggttga ccgtccgtcg gcacaccact tataatgcgg 600 ggtgcaagcg ccgcgtctaa aatttttttt ttttccattt ttgtcgttat tgttatttcc 660 cgttttttgt tttttttgat tttttcggag cgacaaacct ttcgaaacac gtgtcctgaa 720 aattatcctg ggctgcacgt gataatatgt taccctgtcg ggcggcgcct ctttttccct 780 tttctctcac tagtctcttt ttccaatttg ccaccgtgta gcattttgtt gtgctgttac 840 aaccacaaca aaacgaaaaa cccgtatgga catacatata tatatatata tatatatata 900 ttttgttacg cgtgcatttt cttgttgcaa gcagcatgtc taattggtaa ttttaaagct 960 gccaagctct acataaagaa aaacatacat ctatcccgtt 1000

<210> 93

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pSSAl

<400> 93 cactcatatc gggggtctta cttcccatca taaacacgga ccaaagaatt aaattcatca 60 ttcacataag cgaaaaaaag taatcattat tagttaactt ccctttacat gtaaccatag 120 gaaaaaaaaa aaagaaagaa aaacaacgat ctataaagta aactaaatcg gaacataccc 180 ttcaatgtat gacttgtcag ccaaagaagc gtttaattgt ttcaaagttt caatcttgga 240 gaaatcggtg gatgccatta tgtgtgtata tattcggttg gactcttcag tgctgaaagg 300 tagaaagcat gaggtaaaaa gataaagagt agaaggaaaa aaaaaagaac caactaaata 360 tagtgggtag ataaccttaa actatttatt tagaggcgac aatgtgaaaa attttttctt 420 caaaggctcg gttgtcgaca aattgttacg ttgtgctttg atttctaaag cgcttcttca 480 cctgcaggtt ctgagcccta agaaaaaaaa tttccttggt tgaaaatggc ggaaaaaaaa 540 aattcagaaa aagaaataaa gcacgtgtgc gcggtgtgtg gatgatggtt tcatcattgt 600 caacggcatt ttcgttcttg tggattgttg taaactttcc agaacattct agaaagaaag 660 cacacggaac gtttagaagc tgtcatttgc gttttttctc cagattttag ttgagaaagt 720 aattaaatta ttcttctttt tccagaacgt tccatcggcg gcaaaaggga gagaaagaac 780 ccaaaaagaa ggggggccat ttagattagc tgatcgtttc gaggacttca aggttatata 840 aggggtggat tgatgtatct tcgagaaggg attgagttgt agtttcgttt cccaattctt 900 acttaagttg ttttattttc tctatttgta agataagcac atcaaaagaa aagtaatcaa 960 gtattacaag aaacaaaaat tcaagtaaat aacagataat 1000

<210> 94

<211> 1000

<212> DNA

<213> S . cerevisiea

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pFPR2

<400> 94 ctgatctttt cacgcagtta ggtaaggaaa atcgtggcca aataaatttc attgcattaa 60 actctacaat gttcccacac cacgttagat tcctaaatat gagagaacag ttcccattat 120 ttgctatcca taatatgatc aataatctga aatatggttt accacaacta ccagaagaag 180 agtacgcgaa attagaaaaa ccacaaccac tagacagaga tatgatcgtt cagttggtaa 240 aagattaccg tgaaggtact gccaagccaa ttgttaagtc agaagagatt ccaaaagaac 300 aaaagtccaa tgtttataaa atagttggga agacacatga cgacattgtt catgatgatg 360 acaaggatgt ccttgtcaaa tattacgcga catggtgtat tcatagtaaa aggtttgcgc 420 ctatttacga agaaattgca aatgtcttag catctgatga atctgttcgc gataaaatct 480 tgatcgccga agtagattca ggggcaaatg atatcttaag ttttcctgtg acaggatatc 540 caaccattgc tttgtatcct gccggaaata actctaagcc tattatcttc aataaaatta 600 gaaatttgga agatgttttc gaatttatca aggaatcagg tacacatcac attgacggcc 660 aggcaattta tgataaattg caccaggcca aggattctga agtgtctact gaagataccg 720 tacatgatga attataatca ataaataaag catatataat gcacattttt aacatctgat 780 tactcgcatc gtttctggaa gaaaatagct aatattcgtt atttatggca tcacgattat 840 tctcaccagt tacccgttta tgctcttgaa gagatttagc attactgcca gcgcatcttc 900 aaatacaggt ttatatgaga cccattacta taaccctaag aagagaaaaa ggagtgcttt 960 cgttttcaat cacattctag tttacagtaa ttgagtctcg 1000

<210> 95

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pRPL3

<400> 95 cctttcctgc gggatagctt ttgcttcagg ttcttctgag gttctgaaaa tcaatatatt 60 tttccgacaa tttctcacta tgcctctatc ccccttaaaa agaaacactt tctaccctct 120 tttctaaact ctgcacacta gaccgagaca gcaaatcgga gcccgagaga aaatccggag 180 gacggaaaaa ctagaagtaa tcttatgcgg ggagcatgca ccagaacgcc cagaaaacgt 240 gaatgacact acatactttg atacggatgt tggtattttt ccggcttttt ctattttatc 300 gatgccgtgt ctgctgccat tacgtacagg taggcaaagg caaacaagaa tagcactttt 360 tcacttgctg cttttttttg tgaatgaagt ttatcgtata ttctgttagt gccgctcgta 420 tcagccactt acgttatcat tctaaagtaa gtatacgaaa tgtgcaaacc ttagtagacg 480 ttcaagaatt gctcgataat tgcgaacaaa ccagctgcta agccgggaag ttaagtcctc 540 aacacagaaa tgatgcaaac aatttattaa attcagtggt aatgcaacag caagaggaaa 600 ggtggaggga ttaacgcatt tcagacagct ttataggggg aaagaaagca ctcgcaaact 660 tgctgcctgt tcgcagtcat tggttgcaaa aactaaactc tactcacgca cactggaatg 720 aatggcaata ttctttttta ggttaaccgg ccggacagta atatagtaat cgttttgtac 780 gtttttcaag aagcgacgca caactgtttt ccattttttt tttttttttt tcagtgatca 840 tcgtccatga aaaaaatttt tcatttgtct ctttcgtgct tcctggatat ataaaatacg 900 atttatttag ttgtctttgt caatcctcat ctttctttac tcattatttc atttcggttt 960 tgtcatctct agaacaacac agttactaca acaatcaatc 1000

<210> 96

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pRPL6A

<400> 96 attctcgtgc aagcagtttt gcgcgtactt tagctcctca tggcacttac aatggtttta 60 ttaccgtggt tgctgcggaa aacgttgcca agcatgttca aattaagatc tctttaactc 120 aaggtggtag actaaaacac atatacaaaa cgaaaagcca aaaagccaat aatgatggtg 180 ttgccgtatt tgatgaagag tgctcgttca aggcttctcc cgaagccaat ttggtactgg 240 gtgcaatttc ccatcaaaga ctatcgaggg acaaagatct tggtattgct caaatcaact 300 tgggtgaccc tcaaattcaa caagatggcc aaatttctgt aaaattagga gacggtcatc 360 tgattgtaaa gattaattac ggtaaagaca agaatggtca ggtacctccc gtgccagaag 420 ttcctcaaga atacacgcag taataaacag tgaaaactat tcttttatac gctcaataag 480 ggcacatttg gtgtgttaac aggtgtcttt tctgtgtttg ttataatttt gcgctattat 540 tatataacgt tatatttact ataatatatt acaacatata ttacaacata tttcaacgct 600 ctcctgttac tgcttaggga gaagaaagaa atgtgaagac tgctattaaa tccgtacatt 660 attaaaatgt atggatttgt tatatttttc ttcagttttt tccttaacca gattcgaaaa 720 gcggcccgcc tagaccattg cggttgcggc taaagcgttc tggagagggc ttctccgctg 780 ggcggacaac agaagcgaga gacacgtttc ccgtctggga ggactcaccg gatttgtcca 840 gtttcccact ggagtgtgct ttgttccttt tgcaaaattt cgttggtatg gaggttatcg 900 aatggaaggg ttacatgaat agattaaatg tactactcat taaagattga atatcagttt 960 tacattaggc gaaacaacaa agtaatagcc atccaacgaa 1000

<210> 97

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> pRPL28

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pRPL28

<400> 97 gggatagccc tccatgggaa tgtcaaattc tgagcacgaa agcacctgct gaggtgcatt 60 taattccgac aagtggtaac caaattgtat tgaaccgatt gaggctacta taattgcata 120 acataacgcc tttgtaattt tatgcttgtt ttgccttggc atgctaacat tggatagcaa 180 cgatgcacta gatagaatcc tattcatact atgctacgta cctgtttaac tcttctcatt 240 ttatccgttt tctttcttca ccgttcctct taagttcttt atttttttca taacagaatg 300 atcacccctt tcactttgcc gccaatataa tattaacaca caagaaataa gtatgaggta 360 gtttgctctc gaaaaaacca agtaatagtt caaaaaatga aaaaaagaaa acaaatacca 420 aattatgcca ccgttacctt acgtttcatg gttaatcatc gtttactgcc gcctatgagc 480 gtaagctaat gttataaaga aacaagctat aatattgtta aatatagttg atcaacagca 540 ttgtaatgat tacaagagac gaggtggaat gaaccttatg aaatgcgtat tatatataaa 600 ctgtaataag agctaagttg aattgaaatc tacgatactt gatgttgaca ttatagcact 660 agttcccagg aaaccctttc gaaaaacaca gcaaaaacaa gagtactgta accaatgtaa 720 catctgtaca ccagggaccc acacattacc aaaatcaaaa ttatttttct aatgcctgtt 780 atttttccta tttttcctct ggcgcgtgaa tagcccgcag agacgcaaac aattttcctc 840 gcagtttttc gcttgtttaa tgcgtatttt cccagatagg ttcaaacctt tcatctgtat 900 cccgtatatt taagatggcg tttgctttct ccgttgattt ttttccttct tagtgatttt 960 tttgcattaa atcccagaac aatcatccaa ctaatcaaga 1000

<210> 98

<211> 580

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter <222> (1) • • (580)

<223> burden sensing promoter pOGGl

<400> 98 tgcaatcagg ttcttcccaa tcatccgaat cagagagcaa atctaggtct tcttttgaaa 60 gtatggaagg atgcttcaat tgtgcttgaa taaagttatt agacctgaaa ccacgactac 120 tcatagaaaa cgatagtttg gcgtgcgata cgtttttgag taaaagaaaa ctgttgaaac 180 tacagataaa tggccttctt ggtataatat gattcaatgt tgatcttatc cactttggca 240 tgtaaaattg attgatacaa gtaagctaat cgctcaatgg ataatattac aaataaaaat 300 atcgaggcaa taccagtatt tcaatgtctg gccaaataaa tagtttagta cagcaatttg 360 attaacgtta aagttactct cctctttgaa taatgtcgtg gttaaaaagt aaagtatttg 420 tttatacatt aagtgaagaa aattaagacg cgcctttctt taatgtaacg ccaaaaattt 480 tcaagagatg agctgaacag catcacaaaa ctactatttc cagcggaaga aggcatttga 540 agcgtcctga ttcataattg cgattttatt tatcaaccag 580

<210> 99

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pRADSl

<400> 99 ccgcaataaa gggcttcccg gacttggaat taatacctgc taccaccggc cccacaaagt 60 atggaccgaa tcttctttca taaagtgaac ttgacactaa ttgggtaaac gtttcaggct 120 caatggccct ttcttccttc aacttatata ggttcgtctt gtagcggaac atctcgttca 180 gagtagtcac gtcggtagcc agaccggtaa tccctaagaa cacatgccca tagtggaaga 240 ttttttcaaa cttgttcgac acacctagtg actggcttcc gagacgcaaa tcacaggcaa 300 tggccacaca gtctttacct gtcatcgcaa cgacgatacc accgttaata gaacttggat 360 ccgacatttt ttttttatgc tttattcact gttcaatatt ttcaccacaa ttcgcaagaa 420 acgcactcta cttcgaaact acggttcaaa cttacttagc agcttcccga tttaattggc 480 ctttctacta tgccataaac tctttcttcc tctcttttca tcgcccctgc atttgcactt 540 ttttgccacc ggcagtgcca tccggtcaca tgactacacc acgttaatag cgatctggct 600 tatcattgtc acagagtaaa ttaaaatgga cggtaaatgt tggaaatgca ccactaccgt 660 tcttcaacca atctagttta gctatcctgc aacaggtggc cttcttgagc attccctgag 720 cattccaacc ggttgtatca gtgttttatc accgtctcac catatcccac gactaggcca 780 cacttcgtta ccctatgcta cgcgtcattt ccgctatttc tgtcctggtt tgtttacagt 840 acgcgtggtg ggaccataaa ggggaatagt ggggactgga gaaaaaattt tctcagttac 900 ttcttctatc ttccgtagtt tccatatact agtagttgag tgtagcgaca aagagcagac 960 gtagttattt gttaaaggcc tactaatttg ttatcgtcat 1000

<210> 100

<211> 1000

<212> DNA

<213> S . cerevisiae

<220>

<221> promoter

<222> (1) . . (1000)

<223> burden sensing promoter pRAD54

<400> 100 tgcgctacgg ttcctgccgc tcgtgggaac cccacgcaaa acatattatt cgcttctctc 60 tgctgacaac tccggtttac gttataccgt attaggatca ctataagggt tccttcggga 120 ggagggggga ggggaagaat gtacatcgtc ataaggcctt tatggtgtga agtgggtttt 180 gcgtggaaaa ttcgttttca atgatataga gcccacgcat atacgtacat actagtggcc 240 aaaagcgtgg ggtgggcgga caaagctaca ctggtaaaat acaggattct atgaacaata 300 acaacaacca gctcacgttg ctgaacagcc gaggtcagcc gatgcaaccg aggtttccaa 360 agtagcattt ctgtgctagc tatgtctgta ggtttacatt taatggtgcg tggttccagc 420 ttcatgtgct tgcatgtgat gtcctgcaga tggtaagaag attctgaaag ccgcgctagg 480 agaaaaatat tctgctcgaa gatctgtcct cttaagtaga aagcgtgaaa ttgttgcgtt 540 cttgcattac tactcaacgc gtacgcaaat gcgtctactg cacctgcatg ataaagctta 600 tgtatcaaaa atttaacatc ttgaaaatac acaagtggtg caaagatgtg tcacgttctg 660 gacctgagtg gtgccatgta tgctatttaa catgcaaagg ggaagaccct tccgccttac 720 tgcaataata aaaagtattt tacgcgttac ccaatatagc aaagtttcgc gcaaaaaaaa 780 aaataaaaaa caattacaaa caaaaagaaa aaaaaggaaa taatagaaga tctaactgaa 840 gcgaaggcca aaactcttct cacttgacgt aatagccgat acaaaatcta gagcagcaac 900 ttttctcttt cttcactaaa gctgctacga aagtatagaa aaatcaaacg ctcagaactt 960 agctctattt caaggtacca tatatatttc cttataactg 1000

<210> 101

<211> 5776

<212> DNA

<213> synthetic

<220> <221> misc_feature

<222> (1) . . (5776)

<223> plasmid pEG34 <220> <221> CDS <222> (463) . . (1758)

<223> hGH-glycine linker-GFP <400> 101 atcgatgcat gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 60 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 120 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 180 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 240 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg 300 aggtcgacgg tatcgataag cttgatatca gctgaattca ttaaagagga gaaaggtacc 360 atgggtctgg gcaagaaaaa gaaactgctg ctgctgttac tgagcagcgc agttgcagca 420 agctttatga gcctgaccat tagcctgcct ggtgttcagg ca ttt ccg acc att 474

Phe Pro Thr Ile

1 ccg ctg agc cgt ctg ttt gat aat gca atg ctg cgt gca cat cgt ctg 522

Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu

5 10 15 20 cat cag ctg gca ttt gat acc tat caa gaa ttt gaa gaa gcg tat atc 570

His Gin Leu Ala Phe Asp Thr Tyr Gin Glu Phe Glu Glu Ala Tyr Ile

25 30 35 ccg aaa gag cag aaa tat agc ttc ctg cag aat ccg cag acc agc ctg 618

Pro Lys Glu Gin Lys Tyr Ser Phe Leu Gin Asn Pro Gin Thr Ser Leu

40 45 50 tgt ttt agc gaa agc att ccg aca ccg agc aat cgt gaa gaa acc cag 666

Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gin

55 60 65 cag aaa agc aat ctg gaa ctg ctg cgt att agt ctg ctg ctg att cag 714

Gin Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gin

70 75 80 agc tgg ctg gaa ccg gtg cag ttt ctg cgt agc gtt ttt gca aat agc 762

Ser Trp Leu Glu Pro Val Gin Phe Leu Arg Ser Val Phe Ala Asn Ser

85 90 95 100 ctg gtt tat ggt gca agc gat agc aat gtt tat gat ctg ctg aaa gat 810

Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp

105 110 115 ctg gaa gaa ggt att cag acc ctg atg ggt cgt ctg gaa gat ggt tca 858

Leu Glu Glu Gly Ile Gin Thr Leu Met Gly Arg Leu Glu Asp Gly Ser

120 125 130 ccg cgt acc ggt cag atc ttt aaa cag acc tat agc aaa ttc gat acc 906

Pro Arg Thr Gly Gin Ile Phe Lys Gin Thr Tyr Ser Lys Phe Asp Thr

135 140 145 aac agc cat aat gat gat gcc ctg ctg aaa aac tat ggt ctg ctg tat 954 Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr 150 155 160 tgt ttc cgc aaa gat atg gat aaa gtg gaa acc ttt ctg cgc att gtt 1002

Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val

165 170 175 180 cag tgt cgt agc gtt gaa ggt agc tgt ggt ttt ggt ggc atg cgt aaa 1050

Gin Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly Gly Met Arg Lys

185 190 195 ggc gaa gag ctg ttc act ggt ttc gtc act att ctg gtg gaa ctg gat 1098

Gly Glu Glu Leu Phe Thr Gly Phe Val Thr Ile Leu Val Glu Leu Asp

200 205 210 ggt gat gtc aac ggt cat aag ttt tcc gtg cgt ggc gag ggt gaa ggt 1146

Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly

215 220 225 gac gca act aat ggt aaa ctg acg ctg aag ttc atc tgt act act ggt 1194

Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly

230 235 240 aaa ctg ccg gta cct tgg ccg act ctg gta acg acg ctg act tat ggt 1242

Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly

245 250 255 260 gtt cag tgc ttt gct cgt tat ccg gac cac atg aag cag cat gac ttc 1290 Val Gin Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gin His Asp Phe

265 270 275 ttc aag tcc gcc atg ccg gaa ggc tat gtg cag gaa cgc acg att tcc 1338

Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg Thr Ile Ser

280 285 290 ttt aag gat gac ggc acg tac aaa acg cgt gcg gaa gtg aaa ttt gaa 1386

Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu

295 300 305 ggc gat acc ctg gta aac cgc att gag ctg aaa ggc att gac ttt aaa 1434

Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys

310 315 320 gaa gac ggc aat atc ctg ggc cat aag ctg gaa tac aat ttt aac agc 1482

Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser

325 330 335 340 cac aat gtt tac atc acc gcc gat aaa caa aaa aat ggc att aaa gcg 1530

His Asn Val Tyr Ile Thr Ala Asp Lys Gin Lys Asn Gly Ile Lys Ala

345 350 355 aat ttt aaa att cgc cac aac gtg gag gat ggc agc gtg cag ctg gct 1578

Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gin Leu Ala

360 365 370 gat cac tac cag caa aac act cca atc ggt gat ggt cct gtt ctg ctg 1626

Asp His Tyr Gin Gin Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu

375 380 385 cca gac aat cac tat ctg agc acg caa agc gtt ctg tct aaa gat ccg 1674

Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Val Leu Ser Lys Asp Pro 390 395 400 aac gag aaa cgc gat cac atg gtt ctg ctg gag ttc gta acc gca gcg 1722 Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 405 410 415 420 ggc atc acg cat ggt atg gat gaa ctg tac aaa taa gtcgacctgc 1768 Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

425 430 aggcatgcaa gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta 1828 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg 1888 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 1948 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 2008 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca 2068 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga 2128 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt 2188 tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct 2248 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 2308 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 2368 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cagcaaacta 2428 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2488 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2548 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2608 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 2668 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 2728 gtttactcat atatacttta gattgattta cgcgccctgt agcggcgcat taagcgcggc 2788 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 2848 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 2908 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2968 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 3028 gacgttggag tccacgttct ttaatagtgg actcttgttc caaacttgaa caacactcaa 3088 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 3148 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 3208 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 3268 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 3328 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 3388 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3448 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 3508 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gggcatttga 3568 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata 3628 agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt 3688 ctgccattca tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc 3748 aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt 3808 cattaagcat tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca 3868 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga 3928 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg 3988 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt 4048 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac 4108 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac 4168 tatcccatat caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca 4228 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg 4288 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg 4348 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc 4408 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa 4468 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat 4528 caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag 4588 gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt 4648 cgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg 4708 gcttctgttt ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc 4768 aaaagcaccg ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg 4828 atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca 4888 gcagaatatg tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt 4948 cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc 5008 caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc 5068 cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca 5128 ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct 5188 gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct 5248 gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt 5308 tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca 5368 tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt 5428 catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc 5488 agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg 5548 cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc 5608 atcttattaa tcagataaaa tatttgctca tgagcccgaa gtggcgagcc cgatcttccc 5668 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg 5728 ccacgatgcg tccggcgtag aggatctgct catgtttgac agcttatc 5776

<210> 102

<211> 431

<212> PRT

<213> synthetic

<400> 102

Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15

Ala His Arg Leu His Gin Leu Ala Phe Asp Thr Tyr Gin Glu Phe Glu 20 25 30

Glu Ala Tyr Ile Pro Lys Glu Gin Lys Tyr Ser Phe Leu Gin Asn Pro 35 40 45

Gin Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60

Glu Glu Thr Gin Gin Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80

Leu Leu Ile Gin Ser Trp Leu Glu Pro Val Gin Phe Leu Arg Ser Val 85 90 95

Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110

Leu Leu Lys Asp Leu Glu Glu Gly Ile Gin Thr Leu Met Gly Arg Leu 115 120 125

Glu Asp Gly Ser Pro Arg Thr Gly Gin Ile Phe Lys Gin Thr Tyr Ser

130 135 140

Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160

Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

165 170 175

Leu Arg Ile Val Gin Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190

Gly Met Arg Lys Gly Glu Glu Leu Phe Thr Gly Phe Val Thr Ile Leu 195 200 205 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly

210 215 220

Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile 225 230 235 240

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

245 250 255

Leu Thr Tyr Gly Val Gin Cys Phe Ala Arg Tyr Pro Asp His Met Lys 260 265 270

Gin His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu 275 280 285

Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu

290 295 300 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 305 310 315 320 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

325 330 335

Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gin Lys Asn 340 345 350

Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser 355 360 365 Val Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro Ile Gly Asp Gly

370 375 380

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Val Leu 385 390 395 400 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

405 410 415 Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 420 425 430

<210> 103

<211> 660

<212> DNA

<213> synthetic

<220>

<221> CDS

<222> (1) . . ( 660)

<223> Chloramphenicol_acctyltransf erase

<400> 103 atg gag aaa aaa atc act gga tat acc acc gtt gat ata tcc caa tgg 48 Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gin Trp 1 5 10 15 cat cgt aaa gaa cat ttt gag gca ttt cag tca gtt gct caa tgt acc 96 His Arg Lys Glu His Phe Glu Ala Phe Gin Ser Val Ala Gin Cys Thr 20 25 30 tat aac cag acc gtt cag ctg gat att acg gcc ttt tta aag acc gta 144 Tyr Asn Gin Thr Val Gin Leu Asp Ile Thr Ala Phe Leu Lys Thr Val 35 40 45 aag aaa aat aag cac aag ttt tat ccg gcc ttt att cac att ctt gcc 192

Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His Ile Leu Ala

50 55 60 cgc ctg atg aat gct cat ccg gaa ttc cgt atg gca atg aaa gac ggt 240 Arg Leu Met Asn Ala His Pro Glu Phe Arg Met Ala Met Lys Asp Gly 65 70 75 80 gag ctg gtg ata tgg gat agt gtt cac cct tgt tac acc gtt ttc cat 288 Glu Leu Val Ile Trp Asp Ser Val His Pro Cys Tyr Thr Val Phe His 85 90 95 gag caa act gaa acg ttt tca tcg ctc tgg agt gaa tac cac gac gat 336 Glu Gin Thr Glu Thr Phe Ser Ser Leu Trp Ser Glu Tyr His Asp Asp 100 105 110 ttc egg cag ttt cta cac ata tat tcg caa gat gtg gcg tgt tac ggt 384

Phe Arg Gin Phe Leu His Ile Tyr Ser Gin Asp Val Ala Cys Tyr Gly 115 120 125 gaa aac ctg gcc tat ttc cct aaa ggg ttt att gag aat atg ttt ttc 432

Glu Asn Leu Ala Tyr Phe Pro Lys Gly Phe Ile Glu Asn Met Phe Phe

130 135 140 gtc tca gcc aat ccc tgg gtg agt ttc acc agt ttt gat tta aac gtg 480 Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu Asn Val 145 150 155 160 gcc aat atg gac aac ttc ttc gcc ccc gtt ttc acc atg ggc aaa tat 528 Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly Lys Tyr 165 170 175 tat acg caa ggc gac aag gtg ctg atg ccg ctg gcg att cag gtt cat 576

Tyr Thr Gin Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gin Val His 180 185 190 cat gcc gtc tgt gat ggc ttc cat gtc ggc aga atg ctt aat gaa tta 624 His Ala Val Cys Asp Gly Phe His Val Gly Arg Met Leu Asn Glu Leu 195 200 205 caa cag tac tgc gat gag tgg cag ggc ggg gcg taa 660

Gin Gin Tyr Cys Asp Glu Trp Gin Gly Gly Ala 210 215

<210> 104

<211> 219

<212> PRT

<213> synthetic

<400> 104

Met Glu Lys Lys Ile Thr Gly Tyr Thr Thr Val Asp Ile Ser Gin Trp

1 5 10 15

His Arg Lys Glu His Phe Glu Ala Phe Gin Ser Val Ala Gin Cys Thr 20 25 30

Tyr Asn Gin Thr Val Gin Leu Asp Ile Thr Ala Phe Leu Lys Thr Val 35 40 45

Lys Lys Asn Lys His Lys Phe Tyr Pro Ala Phe Ile His Ile Leu Ala 50 55 60

Arg Leu Met Asn Ala His Pro Glu Phe Arg Met Ala Met Lys Asp Gly 65 70 75 80

Glu Leu Val Ile Trp Asp Ser Val His Pro Cys Tyr Thr Val Phe His 85 90 95

Glu Gin Thr Glu Thr Phe Ser Ser Leu Trp Ser Glu Tyr His Asp Asp 100 105 110

Phe Arg Gin Phe Leu His Ile Tyr Ser Gin Asp Val Ala Cys Tyr Gly 115 120 125

Glu Asn Leu Ala Tyr Phe Pro Lys Gly Phe Ile Glu Asn Met Phe Phe

130 135 140 Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu Asn Val 145 150 155 160

Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly Lys Tyr

165 170 175 Tyr Thr Gin Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gin Val His 180 185 190

His Ala Val Cys Asp Gly Phe His Val Gly Arg Met Leu Asn Glu Leu 195 200 205

Gin Gin Tyr Cys Asp Glu Trp Gin Gly Gly Ala

210 215

<210> 105

<211> 4347

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4347 )

<223> plasmid pEGO

<400> 105 atcgatgcat gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 60 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 120 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 180 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 240 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg 300 aggtcgacgg tatcgataag cttgatatcg aattcctgca ggcatgcaag cttggctgtt 360 ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc 420 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga 480 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag 540 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 600 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg 660 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 720 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 780 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 840 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 900 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 960 agtaaaagat gctgaagatc agttgggtgc agcaaactat taactggcga actacttact 1020 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 1080 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 1140 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 1200 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 1260 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 1320 attgatttac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 1380 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 1440 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 1500 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 1560 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 1620 taatagtgga ctcttgttcc aaacttgaac aacactcaac cctatctcgg gctattcttt 1680 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 1740 aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttaaaagg atctaggtga 1800 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1860 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1920 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1980 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 2040 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 2100 acctcgctct gctaatcctg ttaccagtgg ggcatttgag aagcacacgg tcacactgct 2160 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg 2220 ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat ccgcttatta 2280 tcacttattc aggcgtagca ccaggcgttt aagggcacca ataactgcct taaaaaaatt 2340 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat 2400 ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc 2460 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca 2520 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct 2580 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat 2640 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt 2700 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac 2760 cgtctttcat tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa 2820 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat 2880 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt 2940 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt 3000 tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta 3060 tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaaa 3120 agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat tctgcgaagt 3180 gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct gccaacttac 3240 tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc tatcagctgt 3300 ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag 3360 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 3420 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 3480 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 3540 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 3600 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 3660 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 3720 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 3780 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 3840 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 3900 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 3960 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 4020 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 4080 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 4140 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat 4200 atttgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat 4260 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga 4320 ggatctgctc atgtttgaca gcttatc 4347

<210> 106

<211> 6076

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . ( 6076)

<223> Plasmid pENDU5CAM

<220>

<221> CDS

<222> (108) . . ( 911)

<223> ompA-lysostaphin <400> 106 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat 60 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacat atg aaa aag 116

Met Lys Lys 1 aca gct atc gcg att gca gtg gca ctg gct ggt ttc gct acc gta gcg 164

Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala 5 10 15 cag gct gca gca acc cat gaa cat agc gca cag tgg ctg aat aat tac 212

Gin Ala Ala Ala Thr His Glu His Ser Ala Gin Trp Leu Asn Asn Tyr

20 25 30 35 aaa aaa ggt tat ggc tat ggt ccg tat ccg ctg ggt att aat ggt ggt 260

Lys Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly

40 45 50 atg cat tat ggt gtg gat ttc ttt atg aat att ggc aca ccg gtt aaa 308

Met His Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys

55 60 65 gca att agc agc ggt aaa att gtt gaa gca ggt tgg agc aat tat ggt 356

Ala Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly

70 75 80 ggt ggt aat cag att ggc ctg att gaa aat gat ggt gtt cat cgt cag 404

Gly Gly Asn Gin Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gin

85 90 95 tgg tat atg cat ctg agc aaa tac aat gtg aaa gtg ggc gat tat gtt 452

Trp Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val

100 105 110 115 aaa gcc ggt cag att att ggt tgg agc ggt agc acc ggt tat agt acc 500

Lys Ala Gly Gin Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr

120 125 130 gca ccg cat ctg cat ttt cag cgt atg gtt aat agc ttt agc aat agc 548

Ala Pro His Leu His Phe Gin Arg Met Val Asn Ser Phe Ser Asn Ser

135 140 145 acc gca cag gat ccg atg ccg ttt ctg aaa agc gca ggt tat ggt aaa 596

Thr Ala Gin Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys

150 155 160 gca ggc ggt aca gtt acc ccg aca ccg aat acc ggt tgg aaa acc aac 644

Ala Gly Gly Thr Val Thr Pro Thr Pro Asn Thr Gly Trp Lys Thr Asn

165 170 175 aaa tat ggc acc ctg tat aaa agc gaa agc gca agc ttt acc ccg aat 692

Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Pro Asn

180 185 190 195 aca gat att atc acc cgt acc acc ggt ccg ttt cgt agc atg ccg cag 740

Thr Asp Ile Ile Thr Arg Thr Thr Gly Pro Phe Arg Ser Met Pro Gin

200 205 210 agc ggt gtt ctg aaa gca ggt cag acc att cat tat gat gaa gtg atg 788

Ser Gly Val Leu Lys Ala Gly Gin Thr Ile His Tyr Asp Glu Val Met

215 220 225 aaa cag gat ggt cat gtt tgg gtt ggt tat acc ggt aat agc ggt cag 836 Lys Gin Asp Gly His Val Trp Val Gly Tyr Thr Gly Asn Ser Gly Gin 230 235 240 cgt att tat ctg ccg gtt cgt acc tgg aat aaa agc acc aat aca ctg 884 Arg Ile Tyr Leu Pro Val Arg Thr Trp Asn Lys Ser Thr Asn Thr Leu 245 250 255 ggt gtt ctg tgg ggc acc atc aaa taa tgagatccgg ctgctaacaa 931 Gly Val Leu Trp Gly Thr Ile Lys 260 265 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct 991 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt 1051 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 1111 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 1171 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 1231 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 1291 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 1351 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 1411 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 1471 caaaaattta acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt 1531 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 1591 ccgctcaata actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt 1651 gtaattcatt aagcattctg ccgacatgga agccatcaca gacggcatga tgaacctgaa 1711 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg 1771 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg 1831 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt 1891 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt 1951 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt 2011 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag 2071 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct 2131 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag 2191 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg 2251 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact 2311 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt 2371 gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga 2431 caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa 2491 gtgcgtcatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2551 accccgtaga aaagattgtt catgaccaaa atcccttaac gtgatcaaag gatcttcttg 2611 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2671 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2731 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 2791 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2851 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2911 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2971 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 3031 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 3091 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 3151 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 3211 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 3271 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 3331 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg 3391 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt 3451 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 3511 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 3571 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 3631 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt 3691 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca 3751 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt 3811 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga 3871 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 3931 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 3991 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 4051 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 4111 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 4171 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 4231 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg 4291 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga 4351 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg 4411 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta 4471 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc 4531 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 4591 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 4651 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 4711 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc 4771 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga 4831 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg 4891 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt 4951 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc 5011 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct 5071 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc 5131 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga 5191 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc 5251 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg 5311 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga 5371 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg 5431 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc 5491 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt 5551 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt 5611 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg 5671 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc 5731 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg 5791 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag 5851 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc 5911 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg 5971 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt 6031 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcga 6076

<210> 107

<211> 267

<212> PRT

<213> synthetic <400> 107

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15

Thr Val Ala Gin Ala Ala Ala Thr His Glu His Ser Ala Gin Trp Leu

20 25 30

Asn Asn Tyr Lys Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile 35 40 45

Asn Gly Gly Met His Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr 50 55 60

Pro Val Lys Ala Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser 65 70 75 80

Asn Tyr Gly Gly Gly Asn Gin Ile Gly Leu Ile Glu Asn Asp Gly Val 85 90 95

His Arg Gin Trp Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly

100 105 110

Asp Tyr Val Lys Ala Gly Gin Ile Ile Gly Trp Ser Gly Ser Thr Gly 115 120 125

Tyr Ser Thr Ala Pro His Leu His Phe Gin Arg Met Val Asn Ser Phe

130 135 140

Ser Asn Ser Thr Ala Gin Asp Pro Met Pro Phe Leu Lys Ser Ala Gly 145 150 155 160

Tyr Gly Lys Ala Gly Gly Thr Val Thr Pro Thr Pro Asn Thr Gly Trp 165 170 175

Lys Thr Asn Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe

180 185 190

Thr Pro Asn Thr Asp Ile Ile Thr Arg Thr Thr Gly Pro Phe Arg Ser 195 200 205

Met Pro Gin Ser Gly Val Leu Lys Ala Gly Gin Thr Ile His Tyr Asp

210 215 220

Glu Val Met Lys Gin Asp Gly His Val Trp Val Gly Tyr Thr Gly Asn 225 230 235 240 Ser Gly Gin Arg Ile Tyr Leu Pro Val Arg Thr Trp Asn Lys Ser Thr

245 250 255

Asn Thr Leu Gly Val Leu Trp Gly Thr Ile Lys 260 265

<210> 108

<211> 8593

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (8593)

<223> Plasmid pMevT

<220>

<221> CDS

<222> (380) . . (1564 )

<223> Acet yl-CoA_acctyltransf erase

<220>

<221> CDS

<222> (1578) . . (3053)

<223> 3-hydroxy-3-methylglutaryl-coenzyme_A_synthase

<220>

<221> CDS

<222> (3067 ) . . (4575)

<223> N-Terminally_Truncatcd_3-Hydroxy-3-Methylglutaryl_Coenzyme_A_Redu ctase

<400> 108 atcgatgeat gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 60 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 120 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 180 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 240 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg 300 aggtcgaegg tatcgataag cttgatatcg aattcctgca gcccggggat cctctagagt 360 cgactaggag gaatataaa atg aaa aat tgt gtc atc gtc agt gcg gta cgt 412 Met Lys Asn Cys Val Ile Val Ser Ala Val Arg 1 5 10 act gct atc ggt agt ttt aac ggt tca ctc gct tcc acc agc gcc atc 460 Thr Ala Ile Gly Ser Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile 15 20 25 gac ctg ggg gcg aca gta att aaa gcc gcc att gaa cgt gca aaa atc 508 Asp Leu Gly Ala Thr Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile 30 35 40 gat tca caa cac gtt gat gaa gtg att atg ggt aac gtg tta caa gcc 556 Asp Ser Gin His Val Asp Glu Val Ile Met Gly Asn Val Leu Gin Ala 45 50 55 ggg ctg ggg caa aat ccg gcg cgt cag gca ctg tta aaa agc ggg ctg 604

Gly Leu Gly Gin Asn Pro Ala Arg Gin Ala Leu Leu Lys Ser Gly Leu

60 65 70 75 gca gaa acg gtg tgc gga ttc acg gtc aat aaa gta tgt ggt tcg ggt 652

Ala Glu Thr Val Cys Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly

80 85 90 ctt aaa agt gtg gcg ctt gcc gcc cag gcc att cag gca ggt cag gcg 700

Leu Lys Ser Val Ala Leu Ala Ala Gin Ala Ile Gin Ala Gly Gin Ala

95 100 105 cag agc att gtg gcg ggg ggt atg gaa aat atg agt tta gcc ccc tac 748

Gin Ser Ile Val Ala Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr

110 115 120 tta ctc gat gca aaa gca cgc tct ggt tat cgt ctt gga gac gga cag 796

Leu Leu Asp Ala Lys Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gin

125 130 135 gtt tat gac gta atc ctg cgc gat ggc ctg atg tgc gcc acc cat ggt 844 Val Tyr Asp Val Ile Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly

140 145 150 155 tat cat atg ggg att acc gcc gaa aac gtg gct aaa gag tac gga att 892

Tyr His Met Gly Ile Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile

160 165 170 acc cgt gaa atg cag gat gaa ctg gcg cta cat tca cag cgt aaa gcg 940

Thr Arg Glu Met Gin Asp Glu Leu Ala Leu His Ser Gin Arg Lys Ala

175 180 185 gca gcc gca att gag tcc ggt gct ttt aca gcc gaa atc gtc ccg gta 988

Ala Ala Ala Ile Glu Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val

190 195 200 aat gtt gtc act cga aag aaa acc ttc gtc ttc agt caa gac gaa ttc 1036

Asn Val Val Thr Arg Lys Lys Thr Phe Val Phe Ser Gin Asp Glu Phe

205 210 215 ccg aaa gcg aat tca acg gct gaa gcg tta ggt gca ttg cgc ccg gcc 1084

Pro Lys Ala Asn Ser Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala

220 225 230 235 ttc gat aaa gca gga aca gtc acc gct ggg aac gcg tct ggt att aac 1132

Phe Asp Lys Ala Gly Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn

240 245 250 gac ggt gct gcc gct ctg gtg att atg gaa gaa tct gcg gcg ctg gca 1180

Asp Gly Ala Ala Ala Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala

255 260 265 gca ggc ctt acc ccc ctg gct cgc att aaa agt tat gcc agc ggt ggc 1228

Ala Gly Leu Thr Pro Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly

270 275 280 gtg ccc ccc gca ttg atg ggt atg ggg cca gta cct gcc acg caa aaa 1276 Val Pro Pro Ala Leu Met Gly Met Gly Pro Val Pro Ala Thr Gin Lys

285 290 295 gcg tta caa ctg gcg ggg ctg caa ctg gcg gat att gat ctc att gag 1324

Ala Leu Gin Leu Ala Gly Leu Gin Leu Ala Asp Ile Asp Leu Ile Glu 300 305 310 315 gct aat gaa gca ttt gct gca cag ttc ctt gcc gtt ggg aaa aac ctg 1372

Ala Asn Glu Ala Phe Ala Ala Gin Phe Leu Ala Val Gly Lys Asn Leu

320 325 330 ggc ttt gat tct gag aaa gtg aat gtc aac ggc ggg gcc atc gcg ctc 1420

Gly Phe Asp Ser Glu Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu

335 340 345 ggg cat cct atc ggt gcc agt ggt gct cgt att ctg gtc aca cta tta 1468

Gly His Pro Ile Gly Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu

350 355 360 cat gcc atg cag gca cgc gat aaa acg ctg ggg ctg gca aca ctg tgc 1516

His Ala Met Gin Ala Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys

365 370 375 att ggc ggc ggt cag gga att gcg atg gtg att gaa egg ttg aat taa 1564 Ile Gly Gly Gly Gin Gly Ile Ala Met Val Ile Glu Arg Leu Asn

380 385 390 ggaggacagc taa atg aaa ctc tca act aaa ctt tgt tgg tgt ggt att 1613

Met Lys Leu Ser Thr Lys Leu Cys Trp Cys Gly Ile 395 400 405 aaa gga aga ctt agg ccg caa aag caa caa caa tta cac aat aca aac 1661

Lys Gly Arg Leu Arg Pro Gin Lys Gin Gin Gin Leu His Asn Thr Asn

410 415 420 ttg caa atg act gaa cta aaa aaa caa aag acc gct gaa caa aaa acc 1709

Leu Gin Met Thr Glu Leu Lys Lys Gin Lys Thr Ala Glu Gin Lys Thr

425 430 435 aga cct caa aat gtc ggt att aaa ggt atc caa att tac atc cca act 1757

Arg Pro Gin Asn Val Gly Ile Lys Gly Ile Gin Ile Tyr Ile Pro Thr

440 445 450 caa tgt gtc aac caa tct gag cta gag aaa ttt gat ggc gtt tct caa 1805

Gin Cys Val Asn Gin Ser Glu Leu Glu Lys Phe Asp Gly Val Ser Gin

455 460 465 470 ggt aaa tac aca att ggt ctg ggc caa acc aac atg tct ttt gtc aat 1853

Gly Lys Tyr Thr Ile Gly Leu Gly Gin Thr Asn Met Ser Phe Val Asn

475 480 485 gac aga gaa gat atc tac tcg atg tcc cta act gtt ttg tct aag ttg 1901

Asp Arg Glu Asp Ile Tyr Ser Met Ser Leu Thr Val Leu Ser Lys Leu

490 495 500 atc aag agt tac aac atc gac acc aac aaa att ggt aga tta gaa gtc 1949 Ile Lys Ser Tyr Asn Ile Asp Thr Asn Lys Ile Gly Arg Leu Glu Val

505 510 515 ggt act gaa act ctg att gac aag tcc aag tct gtc aag tct gtc ttg 1997

Gly Thr Glu Thr Leu Ile Asp Lys Ser Lys Ser Val Lys Ser Val Leu

520 525 530 atg caa ttg ttt ggt gaa aac act gac gtc gaa ggt att gac acg ctt 2045

Met Gin Leu Phe Gly Glu Asn Thr Asp Val Glu Gly Ile Asp Thr Leu

535 540 545 550 aat gcc tgt tac ggt ggt acc aac gcg ttg ttc aac tct ttg aac tgg 2093 Asn Ala Cys Tyr Gly Gly Thr Asn Ala Leu Phe Asn Ser Leu Asn Trp 555 560 565 att gaa tct aac gca tgg gat ggt aga gac gcc att gta gtt tgc ggt 2141 Ile Glu Ser Asn Ala Trp Asp Gly Arg Asp Ala Ile Val Val Cys Gly 570 575 580 gat att gcc atc tac gat aag ggt gcc gca aga cca acc ggt ggt gcc 2189

Asp Ile Ala Ile Tyr Asp Lys Gly Ala Ala Arg Pro Thr Gly Gly Ala

585 590 595 ggt act gtt gct atg tgg atc ggt cct gat gct cca att gta ttt gac 2237

Gly Thr Val Ala Met Trp Ile Gly Pro Asp Ala Pro Ile Val Phe Asp

600 605 610 tct gta aga gct tct tac atg gaa cac gcc tac gat ttt tac aag cca 2285

Ser Val Arg Ala Ser Tyr Met Glu His Ala Tyr Asp Phe Tyr Lys Pro

615 620 625 630 gat ttc acc agc gaa tat cct tac gtc gat ggt cat ttt tca tta act 2333

Asp Phe Thr Ser Glu Tyr Pro Tyr Val Asp Gly His Phe Ser Leu Thr

635 640 645 tgt tac gtc aag gct ctt gat caa gtt tac aag agt tat tcc aag aag 2381

Cys Tyr Val Lys Ala Leu Asp Gin Val Tyr Lys Ser Tyr Ser Lys Lys

650 655 660 gct att tct aaa ggg ttg gtt agc gat ccc gct ggt tcg gat gct ttg 2429

Ala Ile Ser Lys Gly Leu Val Ser Asp Pro Ala Gly Ser Asp Ala Leu

665 670 675 aac gtt ttg aaa tat ttc gac tac aac gtt ttc cat gtt cca acc tgt 2477

Asn Val Leu Lys Tyr Phe Asp Tyr Asn Val Phe His Val Pro Thr Cys

680 685 690 aaa ttg gtc aca aaa tca tac ggt aga tta cta tat aac gat ttc aga 2525

Lys Leu Val Thr Lys Ser Tyr Gly Arg Leu Leu Tyr Asn Asp Phe Arg

695 700 705 710 gcc aat cct caa ttg ttc cca gaa gtt gac gcc gaa tta gct act cgc 2573

Ala Asn Pro Gin Leu Phe Pro Glu Val Asp Ala Glu Leu Ala Thr Arg

715 720 725 gat tat gac gaa tct tta acc gat aag aac att gaa aaa act ttt gtt 2621

Asp Tyr Asp Glu Ser Leu Thr Asp Lys Asn Ile Glu Lys Thr Phe Val

730 735 740 aat gtt gct aag cca ttc cac aaa gag aga gtt gcc caa tct ttg att 2669

Asn Val Ala Lys Pro Phe His Lys Glu Arg Val Ala Gin Ser Leu Ile

745 750 755 gtt cca aca aac aca ggt aac atg tac acc gca tct gtt tat gcc gcc 2717 Val Pro Thr Asn Thr Gly Asn Met Tyr Thr Ala Ser Val Tyr Ala Ala

760 765 770 ttt gca tct cta tta aac tat gtt gga tct gac gac tta caa ggc aag 2765

Phe Ala Ser Leu Leu Asn Tyr Val Gly Ser Asp Asp Leu Gin Gly Lys

775 780 785 790 cgt gtt ggt tta ttt tct tac ggt tec ggt tta gct gca tct cta tat 2813

Arg Val Gly Leu Phe Ser Tyr Gly Ser Gly Leu Ala Ala Ser Leu Tyr

795 800 805 tct tgc aaa att gtt ggt gac gtc caa cat att atc aag gaa tta gat 2861

Ser Cys Lys Ile Val Gly Asp Val Gin His Ile Ile Lys Glu Leu Asp

810 815 820 att act aac aaa tta gcc aag aga atc acc gaa act cca aag gat tac 2909 Ile Thr Asn Lys Leu Ala Lys Arg Ile Thr Glu Thr Pro Lys Asp Tyr

825 830 835 gaa gct gcc atc gaa ttg aga gaa aat gcc cat ttg aag aag aac ttc 2957

Glu Ala Ala Ile Glu Leu Arg Glu Asn Ala His Leu Lys Lys Asn Phe

840 845 850 aaa cct caa ggt tec att gag cat ttg caa agt ggt gtt tac tac ttg 3005

Lys Pro Gin Gly Ser Ile Glu His Leu Gin Ser Gly Val Tyr Tyr Leu

855 860 865 870 acc aac atc gat gac aaa ttt aga aga tct tac gat gtt aaa aaa taa 3053

Thr Asn Ile Asp Asp Lys Phe Arg Arg Ser Tyr Asp Val Lys Lys

875 880 885 ggaggattac act atg gtt tta acc aat aaa aca gtc att tct gga tcg 3102

Met Val Leu Thr Asn Lys Thr Val Ile Ser Gly Ser

890 895 aaa gtc aaa agt tta tca tct gcg caa tcg agc tca tca gga cct tca 3150

Lys Val Lys Ser Leu Ser Ser Ala Gin Ser Ser Ser Ser Gly Pro Ser

900 905 910 tca tct agt gag gaa gat gat tec cgc gat att gaa agc ttg gat aag 3198

Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser Leu Asp Lys

915 920 925 aaa ata cgt cct tta gaa gaa tta gaa gca tta tta agt agt gga aat 3246

Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu Ser Ser Gly Asn

930 935 940 945 aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg gtt att cac ggt 3294

Thr Lys Gin Leu Lys Asn Lys Glu Val Ala Ala Leu Val Ile His Gly

950 955 960 aag tta cct ttg tac gct ttg gag aaa aaa tta ggt gat act acg aga 3342

Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly Asp Thr Thr Arg

965 970 975 gcg gtt gcg gta cgt agg aag gct ctt tca att ttg gca gaa gct cct 3390

Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu Ala Glu Ala Pro

980 985 990 gta tta gca tct gat cgt tta cca tat aaa aat tat gac tac gac cgc 3438 Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr Asp Tyr Asp Arg

995 1000 1005 gta ttt ggc gct tgt tgt gaa aat gtt ata ggt tac atg cct ttg 3483 Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Leu

1010 1015 1020 ccc gtt ggt gtt ata ggc ccc ttg gtt atc gat ggt aca tct tat 3528

Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr Ser Tyr

1025 1030 1035 cat ata cca atg gca act aca gag ggt tgt ttg gta gct tct gcc 3573

His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Ala

1040 1045 1050 atg cgt ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act 3618

Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr

1055 1060 1065 gtt tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc 3663 Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe

1070 1075 1080 cca act ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca 3708

Pro Thr Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser

1085 1090 1095 gaa gag gga caa aac gca att aaa aaa gct ttt aac tct aca tca 3753

Glu Glu Gly Gin Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser

1100 1105 1110 aga ttt gca cgt ctg caa cat att caa act tgt cta gca gga gat 3798

Arg Phe Ala Arg Leu Gin His Ile Gin Thr Cys Leu Ala Gly Asp

1115 1120 1125 tta ctc ttc atg aga ttt aga aca act act ggt gac gca atg ggt 3843

Leu Leu Phe Met Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly

1130 1135 1140 atg aat atg att tct aaa ggt gtc gaa tac tca tta aag caa atg 3888

Met Asn Met Ile Ser Lys Gly Val Glu Tyr Ser Leu Lys Gin Met

1145 1150 1155 gta gaa gag tat ggc tgg gaa gat atg gag gtt gtc tec gtt tct 3933 Val Glu Glu Tyr Gly Trp Glu Asp Met Glu Val Val Ser Val Ser

1160 1165 1170 ggt aac tac tgt acc gac aaa aaa cca gct gcc atc aac tgg atc 3978

Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ala Ala Ile Asn Trp Ile

1175 1180 1185 gaa ggt cgt ggt aag agt gtc gtc gca gaa gct act att cct ggt 4023

Glu Gly Arg Gly Lys Ser Val Val Ala Glu Ala Thr Ile Pro Gly

1190 1195 1200 gat gtt gtc aga aaa gtg tta aaa agt gat gtt tec gca ttg gtt 4068

Asp Val Val Arg Lys Val Leu Lys Ser Asp Val Ser Ala Leu Val

1205 1210 1215 gag ttg aac att gct aag aat ttg gtt gga tct gca atg gct ggg 4113

Glu Leu Asn Ile Ala Lys Asn Leu Val Gly Ser Ala Met Ala Gly

1220 1225 1230 tct gtt ggt gga ttt aac gca cat gca gct aat tta gtg aca gct 4158

Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn Leu Val Thr Ala

1235 1240 1245 gtt ttc ttg gca tta gga caa gat cct gca caa aat gtt gaa agt 4203 Val Phe Leu Ala Leu Gly Gin Asp Pro Ala Gin Asn Val Glu Ser

1250 1255 1260 tcc aac tgt ata aca ttg atg aaa gaa gtg gac ggt gat ttg aga 4248

Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp Leu Arg

1265 1270 1275 att tcc gta tcc atg cca tcc atc gaa gta ggt acc atc ggt ggt 4293 Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly 1280 1285 1290 ggt act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt 4338

Gly Thr Vai Leu Glu Pro Gin Gly Ala Met Leu Asp Leu Leu Gly 1295 1300 1305 gta aga ggc ccg cat get acc get cct ggt ace aac gca cgt caa 4383

Vai Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gin 1310 1315 1320 tta gca aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc 4428

Leu Ala Arg lie Vai Ala Cys Ala Vai Leu Ala Gly Glu Leu Ser 1325 1330 1335 tta tgt get gcc cta gca gcc ggc cat ttg gtt caa agt cat atg 4473

Leu Cys Ala Ala Leu Ala Ala Gly His Leu Vai Gin Ser His Met 1340 1345 1350 acc cac aac agg aaa cct get gaa cca aca aaa cct aac aat ttg 4518

Thr His Asn Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu 1355 1360 1365 gac gcc act gat ata aat cgt ttg aaa gat ggg tcc gtc acc tgc 4563

Asp Ala Thr Asp lie Asn Arg Leu Lys Asp Gly Ser Vai Thr Cys 1370 1375 1380 att aaa tcc taa gtcgacctgc aggcatgcaa gcttggctgt tttggcggat 4615 lie Lys Ser 1385 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac 4675 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag 4735 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc 4795 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt 4855 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg 4915 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt 4975 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt 5035 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 5095 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 5155 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 5215 tgctgaagat cagttgggtg cagcaaacta ttaactggcg aactacttac tctagcttcc 5275 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 5335 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 5395 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 5455 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 5515 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 5575 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 5635 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 5695 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 5755 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 5815 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 5875 actcttgttc caaacttgaa caacactcaa ccctatctcg ggctattctt ttgatttata 5935 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 5995 cgcgaatttt aacaaaatat taacgtttac aatttaaaag gatctaggtg aagatccttt 6055 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 6115 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 6175 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 6235 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 6295 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 6355 tgctaatcct gttaccagtg gggcatttga gaagcacacg gtcacactgc ttccggtagt 6415 caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc 6475 gacgaccggg tcgaatttgc tttcgaattt ctgccattca tccgcttatt atcacttatt 6535 caggcgtagc accaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc 6595 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat 6655 cacagacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat 6715 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat 6775 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc 6835 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta 6895 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct 6955 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca 7015 ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg 7075 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa 7135 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat 7195 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct 7255 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat 7315 ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc 7375 agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg 7435 tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt 7495 gtatgatggt gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt 7555 tcagctactg acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg 7615 agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg 7675 caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc 7735 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta 7795 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg 7855 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga 7915 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 7975 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg 8035 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc 8095 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa 8155 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg 8215 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga 8275 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc 8335 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta 8395 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttgctca 8455 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag 8515 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatctgct 8575 catgtttgac agcttatc 8593

<210> 109

<211> 394

<212> PRT

<213> synthetic

<400> 109

Met Lys Asn Cys Val Ile Val Ser Ala Val Arg Thr Ala Ile Gly Ser 1 5 10 15

Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile Asp Leu Gly Ala Thr 20 25 30 Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile Asp Ser Gin His Val 35 40 45

Asp Glu Val Ile Met Gly Asn Val Leu Gin Ala Gly Leu Gly Gin Asn 50 55 60

Pro Ala Arg Gin Ala Leu Leu Lys Ser Gly Leu Ala Glu Thr Val Cys 65 70 75 80

Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly Leu Lys Ser Val Ala 85 90 95

Leu Ala Ala Gin Ala Ile Gin Ala Gly Gin Ala Gin Ser Ile Val Ala 100 105 110

Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr Leu Leu Asp Ala Lys 115 120 125

Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gin Val Tyr Asp Val Ile

130 135 140

Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly Tyr His Met Gly Ile 145 150 155 160

Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Met Gin

165 170 175

Asp Glu Leu Ala Leu His Ser Gin Arg Lys Ala Ala Ala Ala Ile Glu 180 185 190

Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val Asn Val Val Thr Arg 195 200 205

Lys Lys Thr Phe Val Phe Ser Gin Asp Glu Phe Pro Lys Ala Asn Ser

210 215 220

Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala Phe Asp Lys Ala Gly 225 230 235 240

Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Ala

245 250 255

Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala Ala Gly Leu Thr Pro 260 265 270

Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly Val Pro Pro Ala Leu 275 280 285

Met Gly Met Gly Pro Val Pro Ala Thr Gin Lys Ala Leu Gin Leu Ala

290 295 300

Gly Leu Gin Leu Ala Asp Ile Asp Leu Ile Glu Ala Asn Glu Ala Phe 305 310 315 320 Ala Ala Gin Phe Leu Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu 325 330 335

Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly His Pro Ile Gly

340 345 350

Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu His Ala Met Gin Ala 355 360 365

Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gin 370 375 380

Gly Ile Ala Met Val Ile Glu Arg Leu Asn 385 390

<210> 110

<211> 491

<212> PRT

<213> synthetic

<400> 110

Met Lys Leu Ser Thr Lys Leu Cys Trp Cys Gly Ile Lys Gly Arg Leu

1 5 10 15

Arg Pro Gin Lys Gin Gin Gin Leu His Asn Thr Asn Leu Gin Met Thr 20 25 30

Glu Leu Lys Lys Gin Lys Thr Ala Glu Gin Lys Thr Arg Pro Gin Asn 35 40 45 Val Gly Ile Lys Gly Ile Gin Ile Tyr Ile Pro Thr Gin Cys Val Asn 50 55 60

Gin Ser Glu Leu Glu Lys Phe Asp Gly Val Ser Gin Gly Lys Tyr Thr 65 70 75 80 Ile Gly Leu Gly Gin Thr Asn Met Ser Phe Val Asn Asp Arg Glu Asp 85 90 95 Ile Tyr Ser Met Ser Leu Thr Val Leu Ser Lys Leu Ile Lys Ser Tyr

100 105 110

Asn Ile Asp Thr Asn Lys Ile Gly Arg Leu Glu Val Gly Thr Glu Thr 115 120 125

Leu Ile Asp Lys Ser Lys Ser Val Lys Ser Val Leu Met Gin Leu Phe

130 135 140 Gly Glu Asn Thr Asp Val Glu Gly Ile Asp Thr Leu Asn Ala Cys Tyr 145 150 155 160

Gly Gly Thr Asn Ala Leu Phe Asn Ser Leu Asn Trp Ile Glu Ser Asn

165 170 175

Ala Trp Asp Gly Arg Asp Ala Ile Val Val Cys Gly Asp Ile Ala Ile 180 185 190

Tyr Asp Lys Gly Ala Ala Arg Pro Thr Gly Gly Ala Gly Thr Val Ala 195 200 205

Met Trp Ile Gly Pro Asp Ala Pro Ile Val Phe Asp Ser Val Arg Ala 210 215 220

Ser Tyr Met Glu His Ala Tyr Asp Phe Tyr Lys Pro Asp Phe Thr Ser 225 230 235 240

Glu Tyr Pro Tyr Val Asp Gly His Phe Ser Leu Thr Cys Tyr Val Lys

245 250 255

Ala Leu Asp Gin Val Tyr Lys Ser Tyr Ser Lys Lys Ala Ile Ser Lys 260 265 270

Gly Leu Val Ser Asp Pro Ala Gly Ser Asp Ala Leu Asn Val Leu Lys 275 280 285

Tyr Phe Asp Tyr Asn Val Phe His Val Pro Thr Cys Lys Leu Val Thr 290 295 300

Lys Ser Tyr Gly Arg Leu Leu Tyr Asn Asp Phe Arg Ala Asn Pro Gin 305 310 315 320

Leu Phe Pro Glu Val Asp Ala Glu Leu Ala Thr Arg Asp Tyr Asp Glu

325 330 335

Ser Leu Thr Asp Lys Asn Ile Glu Lys Thr Phe Val Asn Val Ala Lys 340 345 350

Pro Phe His Lys Glu Arg Val Ala Gin Ser Leu Ile Val Pro Thr Asn 355 360 365

Thr Gly Asn Met Tyr Thr Ala Ser Val Tyr Ala Ala Phe Ala Ser Leu 370 375 380

Leu Asn Tyr Val Gly Ser Asp Asp Leu Gin Gly Lys Arg Val Gly Leu 385 390 395 400 Phe Ser Tyr Gly Ser Gly Leu Ala Ala Ser Leu Tyr Ser Cys Lys Ile 405 410 415 Val Gly Asp Val Gin His Ile Ile Lys Glu Leu Asp Ile Thr Asn Lys

420 425 430

Leu Ala Lys Arg Ile Thr Glu Thr Pro Lys Asp Tyr Glu Ala Ala Ile

435 440 445

Glu Leu Arg Glu Asn Ala His Leu Lys Lys Asn Phe Lys Pro Gin Gly

450 455 460

Ser Ile Glu His Leu Gin Ser Gly Val Tyr Tyr Leu Thr Asn Ile Asp 465 470 475 480

Asp Lys Phe Arg Arg Ser Tyr Asp Val Lys Lys 485 490

<210> 111

<211> 502

<212> PRT

<213> synthetic

<400> 111

Met Val Leu Thr Asn Lys Thr Val Ile Ser Gly Ser Lys Val Lys Ser

1 5 10 15

Leu Ser Ser Ala Gin Ser Ser Ser Ser Gly Pro Ser Ser Ser Ser Glu

20 25 30

Glu Asp Asp Ser Arg Asp Ile Glu Ser Leu Asp Lys Lys Ile Arg Pro 35 40 45

Leu Glu Glu Leu Glu Ala Leu Leu Ser Ser Gly Asn Thr Lys Gin Leu

50 55 60

Lys Asn Lys Glu Val Ala Ala Leu Val Ile His Gly Lys Leu Pro Leu 65 70 75 80

Tyr Ala Leu Glu Lys Lys Leu Gly Asp Thr Thr Arg Ala Val Ala Val 85 90 95

Arg Arg Lys Ala Leu Ser Ile Leu Ala Glu Ala Pro Val Leu Ala Ser

100 105 110

Asp Arg Leu Pro Tyr Lys Asn Tyr Asp Tyr Asp Arg Val Phe Gly Ala 115 120 125 Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Leu Pro Val Gly Val Ile

130 135 140

Gly Pro Leu Val Ile Asp Gly Thr Ser Tyr His Ile Pro Met Ala Thr 145 150 155 160

Thr Glu Gly Cys Leu Val Ala Ser Ala Met Arg Gly Cys Lys Ala Ile 165 170 175

Asn Ala Gly Gly Gly Ala Thr Thr Val Leu Thr Lys Asp Gly Met Thr

180 185 190

Arg Gly Pro Val Val Arg Phe Pro Thr Leu Lys Arg Ser Gly Ala Cys 195 200 205

Lys Ile Trp Leu Asp Ser Glu Glu Gly Gin Asn Ala Ile Lys Lys Ala

210 215 220

Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gin His Ile Gin Thr Cys 225 230 235 240

Leu Ala Gly Asp Leu Leu Phe Met Arg Phe Arg Thr Thr Thr Gly Asp 245 250 255

Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu Tyr Ser Leu Lys

260 265 270

Gin Met Val Glu Glu Tyr Gly Trp Glu Asp Met Glu Val Val Ser Val 275 280 285

Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ala Ala Ile Asn Trp Ile

290 295 300

Glu Gly Arg Gly Lys Ser Val Val Ala Glu Ala Thr Ile Pro Gly Asp 305 310 315 320 Val Val Arg Lys Val Leu Lys Ser Asp Val Ser Ala Leu Val Glu Leu 325 330 335

Asn Ile Ala Lys Asn Leu Val Gly Ser Ala Met Ala Gly Ser Val Gly

340 345 350

Gly Phe Asn Ala His Ala Ala Asn Leu Val Thr Ala Val Phe Leu Ala 355 360 365

Leu Gly Gin Asp Pro Ala Gin Asn Val Glu Ser Ser Asn Cys Ile Thr

370 375 380 Leu Met Lys Glu Val Asp Gly Asp Leu Arg Ile Ser Val Ser Met Pro 385 390 395 400

Ser Ile Glu Val Gly Thr Ile Gly Gly Gly Thr Val Leu Glu Pro Gin 405 410 415

Gly Ala Met Leu Asp Leu Leu Gly Val Arg Gly Pro His Ala Thr Ala

420 425 430

Pro Gly Thr Asn Ala Arg Gin Leu Ala Arg Ile Val Ala Cys Ala Val

435 440 445

Leu Ala Gly Glu Leu Ser Leu Cys Ala Ala Leu Ala Ala Gly His Leu

450 455 460 Val Gin Ser His Met Thr His Asn Arg Lys Pro Ala Glu Pro Thr Lys 465 470 475 480

Pro Asn Asn Leu Asp Ala Thr Asp Ile Asn Arg Leu Lys Asp Gly Ser 485 490 495 Val Thr Cys Ile Lys Ser 500

<210> 112

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) . . (32 )

<223> yccV forward primer

<400> 112 attcatatgu ggttataaag acatcaacat gc 32

<210> 113

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) . . ( 90)

<223> yccV reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65) <223> N is A, T, C or G

<400> 113 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt agtcacctcc cgggaaatct 90

<210> 114

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> ycjX forward primer

<400> 114 attcatatgu ttggctgata ctgtaattct tc 32

<210> 115

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> ycjX reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 115 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt tgctgtcctg tgctgctctg 90

<210> 116

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> mutM forward primer

<400> 116 attcatatgu ggcgaaaagt actaagtact ta 32

<210> 117

<211> 90

<212> DNA <213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> mutM reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 117 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt agcatctcca ggaatgaaca 90

<210> 118

<211> 19

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (19)

<223> primer P492

<400> 118 tgcaactctt cttcaacgc 19

<210> 119

<211> 19

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (19)

<223> primer P493

<400> 119 aagaaccagg cgcttctaa 19

<210> 120

<211> 19

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (19)

<223> primer P525

<400> 120 attgatgacc tgatggcac 19 <210> 121

<211> 18

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (18)

<223> primer P526

<400> 121 gatccagact tcgaagcg 18

<210> 122

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (90)

<223> ibpA forward primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 122 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt aatcaatagc tcctgaaatc 90

<210> 123

<211> 31

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (31)

<223> ibpA reverse primer

<400> 123 attcatatgt ttggcctgat gagttatagc g 31

<210> 124

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> grpE forward primer <400> 124 attcatatgu tctccgcgag cgtgccagtt tt 32

<210> 125

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> grpE reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 125 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt gaatttctcc gcgttttttt 90

<210> 126

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> IdhA forward primer

<400> 126 attcatatgu gggaacccac agcccgagcg tc 32

<210> 127

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> IdhA reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 127 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt aagactttct ccagtgatgt 90 <210> 128

<211> 122

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (122 )

<223> ybbN forward primer

<220>

<221> misc_feature

<222> ( 97 ) . . ( 97 )

<223> N is A, T, C or G

<400> 128 attcatatgu cccattcgta ctcgcttcac cgatccccat taegtgagga tggctaaggt 60 ccagtgaagt accctgggca aagagtttca taccttncaa gtggagtcgc tctctgttgt 120 eg 122

<210> 129

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> ybbN reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 129 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt ggagtcgctc tctgttgtcg 90

<210> 130

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> fxsA forward primer

<400> 130 attcatatgu taaacacaca gaatatatgt gg 32

<210> 131 <211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> fxsA reverse primer

<220>

<221> misc_feature

<222> (65) . . (65)

<223> N is A, T, C or G

<400> 131 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt aggtttctcc tgtaatagca 90

<210> 132

<211> 31

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (31)

<223> rrnB forward primer

<400> 132 attcatatgu gcaaacacgc cgccgggtca g 31

<210> 133

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> rrnB reverse primer

<220>

<221> misc_feature

<222> (65) . . (65)

<223> N is A, T, C or G

<400> 133 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt aaaagtttga cgctcaaaga 90

<210> 134

<211> 35

<212> DNA

<213> synthetic <220>

<221> primer_bind

<222> (1) • • (35)

<223> rrnE forward primer

<400> 134 attcatatgu aatgcgcctc cgttgagacg acaac 35

<210> 135

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> rrnE reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 135 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt aaaagtttga tgctcaaaga 90

<210> 136

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> poxB forwrad primer

<400> 136 attcatatgu taggttgtcg ctgcctgccg tg 32

<210> 137

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> poxB reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T , C or G <400> 137 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt ggttctccat ctcctgaatg 90

<210> 138

<211> 32

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (32 )

<223> cspD forward primer

<400> 138 attcatatgu cggttatcgg cagaacgccc tg 32

<210> 139

<211> 90

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • ( 90)

<223> cspD reverse primer

<220>

<221> misc_feature

<222> ( 65) . . ( 65)

<223> N is A, T, C or G

<400> 139 atccccatta cgtgaggatg gctaaggtcc agtgaagtac cctgggcaaa gagtttcata 60 ccttncaagt gcttcgacat ccttcgcaaa 90

<210> 140

<211> 1902

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (1902 )

<223> intcgration sequence s9 pmutM folP

<220>

<221> CDS

<222> (299) . . (1093)

<223> kanR

<220>

<221> promoter

<222> (1533) . . (1832 )

<223> burden sensing promoter p (mutM) <220>

<221> RBS

<222> (1833) . . (1838)

<223> rbs catalog

<220>

<221> misc_feature

<222> (1838) . . (1838)

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (1844 ) . . (1902 )

<223> beginning of folP gene

<400> 140 aagaaccagg cgcttctaac aattctggcg acaatggtag tccaaaggct cctcgtccgg 60 ttgatgaacc gcgtacgccg aacccgggta acaccatgtc agagcagtta ggcgacaagt 120 aagttcccgc atcagatgac tgtatttgta ccgaaaaccc cggggcgtgc tccggggttt 180 tttcttatca attcatacca gggataacat cagcgattgt gtaggctgga gctgcttcga 240 agttcctata ctttctagag aataggaact tcggaatagg aacttcaaga tcccctta 298 tta gaa gaa ctc gtc aag aag gcg ata gaa ggc gat gcg ctg cga atc 346 Leu Glu Glu Leu Val Lys Lys Ala Ile Glu Gly Asp Ala Leu Arg Ile 1 5 10 15 ggg agc ggc gat acc gta aag cac gag gaa gcg gtc agc cca ttc gcc 394 Gly Ser Gly Asp Thr Val Lys His Glu Glu Ala Val Ser Pro Phe Ala 20 25 30 gcc aag ctc ttc agc aat atc acg ggt agc caa cgc tat gtc ctg ata 442 Ala Lys Leu Phe Ser Asn Ile Thr Gly Ser Gin Arg Tyr Val Leu Ile 35 40 45 gcg gtc cgc cac acc cag ccg gcc aca gtc gat gaa tcc aga aaa gcg 490 Ala Val Arg His Thr Gin Pro Ala Thr Val Asp Glu Ser Arg Lys Ala 50 55 60 gcc att ttc cac cat gat att egg caa gca ggc atc gcc atg ggt cac 538 Ala Ile Phe His His Asp Ile Arg Gin Ala Gly Ile Ala Met Gly His 65 70 75 80 gac gag atc ctc gcc gtc ggg cat gcg cgc ctt gag cct ggc gaa cag 586 Asp Glu Ile Leu Ala Val Gly His Ala Arg Leu Glu Pro Gly Glu Gin 85 90 95 ttc ggc tgg cgc gag ccc ctg atg ctc ttc gtc cag atc atc ctg atc 634 Phe Gly Trp Arg Glu Pro Leu Met Leu Phe Val Gin Ile Ile Leu Ile 100 105 110 gac aag acc ggc ttc cat ccg agt acg tgc tcg ctc gat gcg atg ttt 682 Asp Lys Thr Gly Phe His Pro Ser Thr Cys Ser Leu Asp Ala Met Phe 115 120 125 cgc ttg gtg gtc gaa tgg gca ggt agc egg atc aag cgt atg cag ccg 730 Arg Leu Val Val Glu Trp Ala Gly Ser Arg Ile Lys Arg Met Gin Pro 130 135 140 ccg cat tgc atc agc cat gat gga tac ttt ctc ggc agg agc aag gtg 778 Pro His Cys Ile Ser His Asp Gly Tyr Phe Leu Gly Arg Ser Lys Val 145 150 155 160 aga tga cag gag atc ctg ccc egg cac ttc gcc caa tag cag cca gtc 826 Arg Gin Glu Ile Leu Pro Arg His Phe Ala Gin Gin Pro Val

165 170 cct tcc cgc ttc agt gac aac gtc gag cac agc tgc gca agg aac gcc 874 Pro Ser Arg Phe Ser Asp Asn Val Glu His Ser Cys Ala Arg Asn Ala 175 180 185 190 cgt cgt ggc cag cca cga tag ccg cgc tgc ctc gtc ctg cag ttc att 922 Arg Arg Gly Gin Pro Arg Pro Arg Cys Leu Val Leu Gin Phe Ile 195 200 205 cag ggc acc gga cag gtc ggt ctt gac aaa aag aac egg gcg ccc ctg 970 Gin Gly Thr Gly Gin Val Gly Leu Asp Lys Lys Asn Arg Ala Pro Leu 210 215 220 cgc tga cag ccg gaa cac ggc ggc atc aga gca gcc gat tgt ctg ttg 1018 Arg Gin Pro Glu His Gly Gly Ile Arg Ala Ala Asp Cys Leu Leu

225 230 235 tgc cca gtc ata gcc gaa tag cct ctc cac cca agc ggc cgg aga acc 1066 Cys Pro Val Ile Ala Glu Pro Leu His Pro Ser Gly Arg Arg Thr 240 245 250 tgc gtg caa tcc atc ttg ttc aat cat gcgaaacgat cctcatcctg 1113 Cys Val Gin Ser Ile Leu Phe Asn His

255 260 tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc 1173 agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt 1233 egcttgctgt ccataaaacc gcccagtcta gctatcgcca tgtaagccca etgeaagcta 1293 cctgctttct ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca 1353 tccggggtca gcaccgtttc tgcggactgg ctttctaegt gttccgcttc ctttagcagc 1413 ccttgcgccc tgagtgcttg cggcagcgtg agcttcaaaa gcgctctgaa gttcctatac 1473 tttctagaga ataggaactt cgaaetgeag gtcgaeggat ccccggaata ttcatatgtg 1533 gcgaaaagta ctaagtactt agaggaaata aatcatggct aaaggtattc gtgagaaaat 1593 caagctggtt tcttctgctg gtactggtca cttctatacc actacgaaga acaaacgtac 1653 taagceggaa aaactggaac tgaaaaaatt cgatccagtt gttcgccagc aegtgatcta 1713 caaagaagcg aaaatcaaat aattctegct ttgatgtaac aaaaaacctc gctccggcgg 1773 ggttttttgt tatctgcttg cccccatatt gaetgeatct gttcattcct ggagatgcta 1833 cttgnaaggt atgaaactct ttgcccaggg tacttcactg gaccttagcc atcctcacgt 1893 aatggggat 1902

<210> 141

<211> 161 <212> PRT

<213> synthetic

<400> 141

Leu Glu Glu Leu Val Lys Lys Ala Ile Glu Gly Asp Ala Leu Arg Ile

1 5 10 15

Gly Ser Gly Asp Thr Val Lys His Glu Glu Ala Val Ser Pro Phe Ala 20 25 30

Ala Lys Leu Phe Ser Asn Ile Thr Gly Ser Gin Arg Tyr Val Leu Ile 35 40 45

Ala Val Arg His Thr Gin Pro Ala Thr Val Asp Glu Ser Arg Lys Ala 50 55 60

Ala Ile Phe His His Asp Ile Arg Gin Ala Gly Ile Ala Met Gly His 65 70 75 80

Asp Glu Ile Leu Ala Val Gly His Ala Arg Leu Glu Pro Gly Glu Gin 85 90 95

Phe Gly Trp Arg Glu Pro Leu Met Leu Phe Val Gin Ile Ile Leu Ile 100 105 110

Asp Lys Thr Gly Phe His Pro Ser Thr Cys Ser Leu Asp Ala Met Phe 115 120 125

Arg Leu Val Val Glu Trp Ala Gly Ser Arg Ile Lys Arg Met Gin Pro 130 135 140

Pro His Cys Ile Ser His Asp Gly Tyr Phe Leu Gly Arg Ser Lys Val 145 150 155 160

Arg

<210> 142

<211> 10

<212> PRT

<213> synthetic

<400> 142

Gin Glu Ile Leu Pro Arg His Phe Ala Gin

1 5 10

<210> 143

<211> 25

<212> PRT <213> synthetic

<400> 143

Gin Pro Val Pro Ser Arg Phe Ser Asp Asn Val Glu His Ser Cys Ala

1 5 10 15

Arg Asn Ala Arg Arg Gly Gin Pro Arg 20 25

<210> 144

<211> 26

<212> PRT

<213> synthetic

<400> 144

Pro Arg Cys Leu Val Leu Gin Phe Ile Gin Gly Thr Gly Gin Val Gly

1 5 10 15

Leu Asp Lys Lys Asn Arg Ala Pro Leu Arg

20 25

<210> 145

<211> 20

<212> PRT

<213> synthetic

<400> 145

Gin Pro Glu His Gly Gly Ile Arg Ala Ala Asp Cys Leu Leu Cys Pro

1 5 10 15 Val Ile Ala Glu 20

<210> 146

<211> 18

<212> PRT

<213> synthetic

<400> 146

Pro Leu His Pro Ser Gly Arg Arg Thr Cys Val Gin Ser Ile Leu Phe

1 5 10 15

Asn His

<210> 147

<211> 1347

<212> DNA

<213> Bacillus licheniformis <220>

<221> CDS

<222> (1) . . (1347 )

<223> Bacillus licheniformis essential gene glmM

<400> 147 atg ggc aag tat ttt ggt aca gac ggt gta aga ggc gtg gca aac agt 48

Met Gly Lys Tyr Phe Gly Thr Asp Gly Val Arg Gly Val Ala Asn Ser 1 5 10 15 gaa ctt aca cct gag ctg gcc ttt aaa gtc gga cgc ttt ggc gga tat 96

Glu Leu Thr Pro Glu Leu Ala Phe Lys Val Gly Arg Phe Gly Gly Tyr

20 25 30 gtc cta aca aaa gat aag gag cgt ccc aag gtt ctg atc ggc cgt gac 144 Val Leu Thr Lys Asp Lys Glu Arg Pro Lys Val Leu Ile Gly Arg Asp

35 40 45 acg cgc att tca ggg cat atg ctg gaa ggg gcc ctt gta gca ggc ctt 192

Thr Arg Ile Ser Gly His Met Leu Glu Gly Ala Leu Val Ala Gly Leu

50 55 60 ctt tcg ata gga gca gaa gtc atg cgt ctc ggc gtg att tcg acg ccc 240

Leu Ser Ile Gly Ala Glu Val Met Arg Leu Gly Val Ile Ser Thr Pro

65 70 75 80 gga gtc gca tat ttg acg aag gct atg gac gcg gag gcg ggt gtg atg 288

Gly Val Ala Tyr Leu Thr Lys Ala Met Asp Ala Glu Ala Gly Val Met

85 90 95 att tec gct tec cac aac cct gtc cag gac aac gga att aag ttt ttc 336 Ile Ser Ala Ser His Asn Pro Val Gin Asp Asn Gly Ile Lys Phe Phe

100 105 110 ggc ggc gac ggc ttt aag ctt tcc gat gaa cag gag ctt gaa atc gag 384

Gly Gly Asp Gly Phe Lys Leu Ser Asp Glu Gin Glu Leu Glu Ile Glu

115 120 125 cgt ctg atg gat cag ccg gaa gat cac ctg cca agg cct gta ggc gct 432

Arg Leu Met Asp Gin Pro Glu Asp His Leu Pro Arg Pro Val Gly Ala

130 135 140 gat ctg ggc atg gtg aac gac tac ttt gaa ggc gga cag aaa tat ttg 480

Asp Leu Gly Met Val Asn Asp Tyr Phe Glu Gly Gly Gin Lys Tyr Leu

145 150 155 160 cag ttt ctt aag cag tct gct gat gaa gac ttc aca ggc att cat gtc 528

Gin Phe Leu Lys Gin Ser Ala Asp Glu Asp Phe Thr Gly Ile His Val

165 170 175 gcg ctt gac tgt gcg cac ggg gcg aca tct tcg ctg gcg acc cac ctg 576

Ala Leu Asp Cys Ala His Gly Ala Thr Ser Ser Leu Ala Thr His Leu

180 185 190 ttt gcc gat ctt gac gca gat gta tcg acg atg ggg act tct ccg aac 624

Phe Ala Asp Leu Asp Ala Asp Val Ser Thr Met Gly Thr Ser Pro Asn

195 200 205 gga tta aac atc aat gac gga gtc gga tca acc cac cct gaa gcc ctg 672

Gly Leu Asn Ile Asn Asp Gly Val Gly Ser Thr His Pro Glu Ala Leu

210 215 220 gca gaa ttt gtg aag gaa aag gga gct gac gtc ggc atg gcg ttt gac 720 Ala Glu Phe Val Lys Glu Lys Gly Ala Asp Val Gly Met Ala Phe Asp 225 230 235 240 ggg gat ggc gac cgc tta atc gct gtc gac gaa aaa ggg aat atc gta 768

Gly Asp Gly Asp Arg Leu Ile Ala Val Asp Glu Lys Gly Asn Ile Val

245 250 255 gac ggc gat caa atc atg tac ata tgc gcg aag tac ttg aag agc gag 816

Asp Gly Asp Gin Ile Met Tyr Ile Cys Ala Lys Tyr Leu Lys Ser Glu

260 265 270 ggg cgc ctg acg gac aat acc gtt gtc tcc acg gtg atg agc aac ctc 864

Gly Arg Leu Thr Asp Asn Thr Val Val Ser Thr Val Met Ser Asn Leu

275 280 285 ggt ttc tac aaa gcg ctt gaa gcg gaa ggc att aaa agc gtt cag aca 912

Gly Phe Tyr Lys Ala Leu Glu Ala Glu Gly Ile Lys Ser Val Gin Thr

290 295 300 gcg gtc ggc gac cgc tat gta gtg gag gcg atg aaa aag ggc gga ttt 960

Ala Val Gly Asp Arg Tyr Val Val Glu Ala Met Lys Lys Gly Gly Phe

305 310 315 320 acc ctc ggc gga gaa cag tcg gga cac ctg att ttc ctt gat tac aat 1008

Thr Leu Gly Gly Glu Gin Ser Gly His Leu Ile Phe Leu Asp Tyr Asn

325 330 335 acg aca ggc gac gga ctg ctt tct gcg atc atg ctg atg aac acg att 1056

Thr Thr Gly Asp Gly Leu Leu Ser Ala Ile Met Leu Met Asn Thr Ile

340 345 350 aaa atg aca ggc aag ccg ctg tct gag ctt gcg gca gag atg cag aag 1104

Lys Met Thr Gly Lys Pro Leu Ser Glu Leu Ala Ala Glu Met Gin Lys

355 360 365 ttt ccg cag ctc ctc ttg aat gtg aaa gta acc gat aaa cat aaa gta 1152

Phe Pro Gin Leu Leu Leu Asn Val Lys Val Thr Asp Lys His Lys Val

370 375 380 acg gaa aac gaa aag gta aaa gcg gtc atc gaa gaa gtt gag aag gaa 1200

Thr Glu Asn Glu Lys Val Lys Ala Val Ile Glu Glu Val Glu Lys Glu

385 390 395 400 atg aac ggt gac ggc cgc atc ctt gtg cgc cca tcc ggc acc gag ccg 1248

Met Asn Gly Asp Gly Arg Ile Leu Val Arg Pro Ser Gly Thr Glu Pro

405 410 415 ctt gtc cgt gtc atg gct gaa gcg aaa acg aag gag ctt tgc gag aag 1296

Leu Val Arg Val Met Ala Glu Ala Lys Thr Lys Glu Leu Cys Glu Lys

420 425 430 tac gta ggc cgc att gca gat gtc gtg aaa gct gaa atg gga gca gag 1344

Tyr Val Gly Arg Ile Ala Asp Val Val Lys Ala Glu Met Gly Ala Glu

435 440 445 taa 1347

<210> 148

<211> 448

<212> PRT

<213> Bacillus licheniformis <400> 148

Met Gly Lys Tyr Phe Gly Thr Asp Gly Val Arg Gly Val Ala Asn Ser

1 5 10 15

Glu Leu Thr Pro Glu Leu Ala Phe Lys Val Gly Arg Phe Gly Gly Tyr 20 25 30 Val Leu Thr Lys Asp Lys Glu Arg Pro Lys Val Leu Ile Gly Arg Asp 35 40 45

Thr Arg Ile Ser Gly His Met Leu Glu Gly Ala Leu Val Ala Gly Leu 50 55 60

Leu Ser Ile Gly Ala Glu Val Met Arg Leu Gly Val Ile Ser Thr Pro 65 70 75 80

Gly Val Ala Tyr Leu Thr Lys Ala Met Asp Ala Glu Ala Gly Val Met 85 90 95 Ile Ser Ala Ser His Asn Pro Val Gin Asp Asn Gly Ile Lys Phe Phe

100 105 110

Gly Gly Asp Gly Phe Lys Leu Ser Asp Glu Gin Glu Leu Glu Ile Glu 115 120 125

Arg Leu Met Asp Gin Pro Glu Asp His Leu Pro Arg Pro Val Gly Ala

130 135 140

Asp Leu Gly Met Val Asn Asp Tyr Phe Glu Gly Gly Gin Lys Tyr Leu 145 150 155 160

Gin Phe Leu Lys Gin Ser Ala Asp Glu Asp Phe Thr Gly Ile His Val

165 170 175

Ala Leu Asp Cys Ala His Gly Ala Thr Ser Ser Leu Ala Thr His Leu 180 185 190

Phe Ala Asp Leu Asp Ala Asp Val Ser Thr Met Gly Thr Ser Pro Asn 195 200 205

Gly Leu Asn Ile Asn Asp Gly Val Gly Ser Thr His Pro Glu Ala Leu

210 215 220

Ala Glu Phe Val Lys Glu Lys Gly Ala Asp Val Gly Met Ala Phe Asp 225 230 235 240

Gly Asp Gly Asp Arg Leu Ile Ala Val Asp Glu Lys Gly Asn Ile Val 245 250 255

Asp Gly Asp Gin Ile Met Tyr Ile Cys Ala Lys Tyr Leu Lys Ser Glu 260 265 270

Gly Arg Leu Thr Asp Asn Thr Val Val Ser Thr Val Met Ser Asn Leu 275 280 285

Gly Phe Tyr Lys Ala Leu Glu Ala Glu Gly Ile Lys Ser Val Gin Thr

290 295 300

Ala Val Gly Asp Arg Tyr Val Val Glu Ala Met Lys Lys Gly Gly Phe 305 310 315 320

Thr Leu Gly Gly Glu Gin Ser Gly His Leu Ile Phe Leu Asp Tyr Asn

325 330 335

Thr Thr Gly Asp Gly Leu Leu Ser Ala Ile Met Leu Met Asn Thr Ile

340 345 350

Lys Met Thr Gly Lys Pro Leu Ser Glu Leu Ala Ala Glu Met Gin Lys 355 360 365

Phe Pro Gin Leu Leu Leu Asn Val Lys Val Thr Asp Lys His Lys Val

370 375 380

Thr Glu Asn Glu Lys Val Lys Ala Val Ile Glu Glu Val Glu Lys Glu 385 390 395 400

Met Asn Gly Asp Gly Arg Ile Leu Val Arg Pro Ser Gly Thr Glu Pro

405 410 415

Leu Val Arg Val Met Ala Glu Ala Lys Thr Lys Glu Leu Cys Glu Lys 420 425 430

Tyr Val Gly Arg Ile Ala Asp Val Val Lys Ala Glu Met Gly Ala Glu

435 440 445

<210> 149

<211> 282

<212> DNA

<213> Bacillus licheniformis

<220>

<221> CDS

<222> (1) . . (282 )

<223> Bacillus licheniformis essential gene ylaN

<400> 149 ttg gcg tct gag att gta gtc gac cat cgt gaa aaa gca ctt gct etg Leu Ala Ser Glu Ile Val Val Asp His Arg Glu Lys Ala Leu Ala Leu 1 5 10 15 tta aaa cgt gat gct gat aaa atc tta aag ctg att caa gtg caa atg 96

Leu Lys Arg Asp Ala Asp Lys Ile Leu Lys Leu Ile Gin Val Gin Met

20 25 30 gac aac tta acg atg cct caa tgt cct ctt tat gaa gag gtt tta gat 144

Asp Asn Leu Thr Met Pro Gin Cys Pro Leu Tyr Glu Glu Val Leu Asp

35 40 45 aca caa atg ttc gga ctc tct aga gaa att gat ttt gct gtc cgc ctt 192

Thr Gin Met Phe Gly Leu Ser Arg Glu Ile Asp Phe Ala Val Arg Leu

50 55 60 gga tta gtg gat gaa gaa gag ggt aaa gaa ctg ctt tac agg ctg gag 240

Gly Leu Val Asp Glu Glu Glu Gly Lys Glu Leu Leu Tyr Arg Leu Glu

65 70 75 80 cgt gaa ttg tct gca tta cat gat gca ttt aca aaa aaa taa 282

Arg Glu Leu Ser Ala Leu His Asp Ala Phe Thr Lys Lys

85 90

<210> 150

<211> 93

<212> PRT

<213> Bacillus licheniformis

<400> 150

Leu Ala Ser Glu Ile Val Val Asp His Arg Glu Lys Ala Leu Ala Leu

1 5 10 15

Leu Lys Arg Asp Ala Asp Lys Ile Leu Lys Leu Ile Gin Val Gin Met 20 25 30

Asp Asn Leu Thr Met Pro Gin Cys Pro Leu Tyr Glu Glu Val Leu Asp 35 40 45

Thr Gin Met Phe Gly Leu Ser Arg Glu Ile Asp Phe Ala Val Arg Leu 50 55 60

Gly Leu Val Asp Glu Glu Glu Gly Lys Glu Leu Leu Tyr Arg Leu Glu 65 70 75 80

Arg Glu Leu Ser Ala Leu His Asp Ala Phe Thr Lys Lys 85 90

<210> 151

<211> 219

<212> DNA

<213> Bacillus licheniformis

<220> <221> CDS

<222> (1) . . (219)

<223> Bacillus licheniformis essential gene infA

<400> 151 atg gcg aaa gac gat gta att gaa gtg gaa ggt act gta gtc gaa acg 48 Met Ala Lys Asp Asp Val Ile Glu Val Glu Gly Thr Val Val Glu Thr 1 5 10 15 ctg cca aac gca atg ttc aaa gtt gaa ctt gag aat ggc cac acg gtt 96

Leu Pro Asn Ala Met Phe Lys Val Glu Leu Glu Asn Gly His Thr Val

20 25 30 ttg gct cac gta tct ggt aaa atc cgc atg cac ttc att cgc att tta 144

Leu Ala His Val Ser Gly Lys Ile Arg Met His Phe Ile Arg Ile Leu

35 40 45 cct gga gac aaa gtt acg gta gaa tta tct cca tat gac tta act cgt 192

Pro Gly Asp Lys Val Thr Val Glu Leu Ser Pro Tyr Asp Leu Thr Arg

50 55 60 ggt aga att acg tac cgt tac aaa taa 219

Gly Arg Ile Thr Tyr Arg Tyr Lys

65 70

<210> 152

<211> 72

<212> PRT

<213> Bacillus licheniformis

<400> 152

Met Ala Lys Asp Asp Val Ile Glu Val Glu Gly Thr Val Val Glu Thr

1 5 10 15

Leu Pro Asn Ala Met Phe Lys Val Glu Leu Glu Asn Gly His Thr Val 20 25 30

Leu Ala His Val Ser Gly Lys Ile Arg Met His Phe Ile Arg Ile Leu 35 40 45

Pro Gly Asp Lys Val Thr Val Glu Leu Ser Pro Tyr Asp Leu Thr Arg 50 55 60

Gly Arg Ile Thr Tyr Arg Tyr Lys 65 70

<210> 153

<211> 870

<212> DNA

<213> Bacillus licheniformis

<220>

<221> CDS

<222> (1) . . (870)

<223> Bacillus licheniformis essential gene dapA <400> 153 atg aac ttc gga aat atc gca acc gcg atg gtt acg ccc ttt gac aaa 48

Met Asn Phe Gly Asn Ile Ala Thr Ala Met Val Thr Pro Phe Asp Lys

1 5 10 15 aat gaa aat atc gat ttt caa aaa ctg tca aag ctg atc gat tat tta 96

Asn Glu Asn Ile Asp Phe Gin Lys Leu Ser Lys Leu Ile Asp Tyr Leu

20 25 30 cta aac aat ggg acg gat tcc ctg gtc gtt gcc gga aca acc gga gaa 144

Leu Asn Asn Gly Thr Asp Ser Leu Val Val Ala Gly Thr Thr Gly Glu

35 40 45 tcg ccg acg ctt tcc gaa gaa gaa aag gta gcg ctc att caa tac tct 192

Ser Pro Thr Leu Ser Glu Glu Glu Lys Val Ala Leu Ile Gin Tyr Ser

50 55 60 gtc aag gaa gcg gca ggc cgt gcc cca atc atc gcc ggc acg gga agc 240 Val Lys Glu Ala Ala Gly Arg Ala Pro Ile Ile Ala Gly Thr Gly Ser

65 70 75 80 aac aac acg aag gcg tcg atc aag ctg acg aaa aaa gct gaa gaa gcc 288

Asn Asn Thr Lys Ala Ser Ile Lys Leu Thr Lys Lys Ala Glu Glu Ala

85 90 95 ggc gct gac gct gtc atg ctc gtc act ccg tac tac aac aag cct tct 336

Gly Ala Asp Ala Val Met Leu Val Thr Pro Tyr Tyr Asn Lys Pro Ser

100 105 110 cag gaa ggc atg tac cgc cat ttc aga gcg att gcg gag gaa aca tcg 384

Gin Glu Gly Met Tyr Arg His Phe Arg Ala Ile Ala Glu Glu Thr Ser

115 120 125 ctg cct gtc atg ctt tat aat gtg ccg gga aga acg gct gct tcg ctc 432

Leu Pro Val Met Leu Tyr Asn Val Pro Gly Arg Thr Ala Ala Ser Leu

130 135 140 gca cca gag acg aca atc cgc ctt gcg gag atc ccg aat att atc gcc 480

Ala Pro Glu Thr Thr Ile Arg Leu Ala Glu Ile Pro Asn Ile Ile Ala

145 150 155 160 att aaa gaa gcg agc ggc gac cta gat gcg ata aca aaa atc gta gcc 528 Ile Lys Glu Ala Ser Gly Asp Leu Asp Ala Ile Thr Lys Ile Val Ala

165 170 175 gaa acc cct gag gac ttt gca gtt tat tca gga gat gac agc ctg aca 576

Glu Thr Pro Glu Asp Phe Ala Val Tyr Ser Gly Asp Asp Ser Leu Thr

180 185 190 ctg ccg gcg ctg tcc gtg gga gcg aga gga atc gtt tcc gtt gca tca 624

Leu Pro Ala Leu Ser Val Gly Ala Arg Gly Ile Val Ser Val Ala Ser

195 200 205 cac atc atc gga ccg gaa atg cag gaa atg atc aaa cat tat acc gag 672

His Ile Ile Gly Pro Glu Met Gin Glu Met Ile Lys His Tyr Thr Glu

210 215 220 ggg aac acg gct cag gct gct ttg atc cat caa aag ctg ctt ccg ctg 720

Gly Asn Thr Ala Gin Ala Ala Leu Ile His Gin Lys Leu Leu Pro Leu

225 230 235 240 atg aaa ggg ctt ttt gcc gca ccg aac ccg tct cca tta aag acg gct 768 Met Lys Gly Leu Phe Ala Ala Pro Asn Pro Ser Pro Leu Lys Thr Ala 245 250 255 ctt cag ctg aag ggt ctt gac gtc ggg tcc gtc egg ctc cct ttg att 816

Leu Gin Leu Lys Gly Leu Asp Val Gly Ser Val Arg Leu Pro Leu Ile

260 265 270 ccg cta aat gaa gat gaa cga ctc cgt tta agc agc ctg atg aac ggc 864

Pro Leu Asn Glu Asp Glu Arg Leu Arg Leu Ser Ser Leu Met Asn Gly

275 280 285 ctg taa 870

Leu

<210> 154

<211> 289

<212> PRT

<213> Bacillus licheniformis

<400> 154

Met Asn Phe Gly Asn Ile Ala Thr Ala Met Val Thr Pro Phe Asp Lys 1 5 10 15

Asn Glu Asn Ile Asp Phe Gin Lys Leu Ser Lys Leu Ile Asp Tyr Leu 20 25 30

Leu Asn Asn Gly Thr Asp Ser Leu Val Val Ala Gly Thr Thr Gly Glu 35 40 45

Ser Pro Thr Leu Ser Glu Glu Glu Lys Val Ala Leu Ile Gin Tyr Ser 50 55 60 Val Lys Glu Ala Ala Gly Arg Ala Pro Ile Ile Ala Gly Thr Gly Ser 65 70 75 80

Asn Asn Thr Lys Ala Ser Ile Lys Leu Thr Lys Lys Ala Glu Glu Ala 85 90 95

Gly Ala Asp Ala Val Met Leu Val Thr Pro Tyr Tyr Asn Lys Pro Ser 100 105 110

Gin Glu Gly Met Tyr Arg His Phe Arg Ala Ile Ala Glu Glu Thr Ser 115 120 125

Leu Pro Val Met Leu Tyr Asn Val Pro Gly Arg Thr Ala Ala Ser Leu

130 135 140

Ala Pro Glu Thr Thr Ile Arg Leu Ala Glu Ile Pro Asn Ile Ile Ala 145 150 155 160 Ile Lys Glu Ala Ser Gly Asp Leu Asp Ala Ile Thr Lys Ile Val Ala

165 170 175

Glu Thr Pro Glu Asp Phe Ala Val Tyr Ser Gly Asp Asp Ser Leu Thr 180 185 190

Leu Pro Ala Leu Ser Val Gly Ala Arg Gly Ile Val Ser Val Ala Ser 195 200 205

His Ile Ile Gly Pro Glu Met Gin Glu Met Ile Lys His Tyr Thr Glu

210 215 220

Gly Asn Thr Ala Gin Ala Ala Leu Ile His Gin Lys Leu Leu Pro Leu 225 230 235 240

Met Lys Gly Leu Phe Ala Ala Pro Asn Pro Ser Pro Leu Lys Thr Ala

245 250 255

Leu Gin Leu Lys Gly Leu Asp Val Gly Ser Val Arg Leu Pro Leu Ile 260 265 270

Pro Leu Asn Glu Asp Glu Arg Leu Arg Leu Ser Ser Leu Met Asn Gly 275 280 285

Leu

<210> 155

<211> 2830

<212> DNA

<213> synthetic

<220>

<221> promoter

<222> (1) . . (1000)

<223> pAOXl promoter

<220>

<221> misc_feature

<222> (1) . . (2830)

<223> pAOXl-ALB construct

<220>

<221> CDS

<222> (1001) . . (2830)

<223> cds encoding Human_serum_albumin (hAS) protein

<400> 155 atgttggtat tgtgaaatag acgcagatcg ggaacactga aaaataacag ttattattcg 60 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 120 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 180 tgeaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 240 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 300 acaccatgac tttattagce tgtctatcct ggcccccctg gcgaggttca tgtttgttta 360 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 420 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaaegct 480 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 540 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggea taccgtttgt 600 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 660 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 720 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 780 gctgatagce taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 840 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 900 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 960 caacttgaga agatcaaaaa acaactaatt attcgaaacg atg aag tgg gta acc 1015 Met Lys Trp Val Thr 1 5 ttt att tcc ctt ctt ttt ctc ttt agc tcg gct tat tcc agg ggt gtg 1063 Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val 10 15 20 ttt cgt cga gat gca cac aag agt gag gtt gct cat egg ttt aaa gat 1111 Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 25 30 35 ttg gga gaa gaa aat ttc aaa gcc ttg gtg ttg att gcc ttt gct cag 1159 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gin 40 45 50 tat ctt cag cag tgt cca ttt gaa gat cat gta aaa tta gtg aat gaa 1207 Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 55 60 65 gta act gaa ttt gca aaa aca tgt gtt gct gat gag tca gct gaa aat 1255 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 70 75 80 85 tgt gac aaa tca ctt cat acc ctt ttt gga gac aaa tta tgc aca gtt 1303 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 90 95 100 gca act ctt cgt gaa acc tat ggt gaa atg gct gac tgc tgt gca aaa 1351 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 105 110 115 caa gaa cct gag aga aat gaa tgc ttc ttg caa cac aaa gat gac aac 1399 Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin His Lys Asp Asp Asn 120 125 130 cca aac ctc ccc cga ttg gtg aga cca gag gtt gat gtg atg tgc act 1447

Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr

135 140 145 gct ttt cat gac aat gaa gag aca ttt ttg aaa aaa tac tta tat gaa 1495

Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu

150 155 160 165 att gcc aga aga cat cct tac ttt tat gcc ccg gaa ctc ctt ttc ttt 1543 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe

170 175 180 gct aaa agg tat aaa gct gct ttt aca gaa tgt tgc caa gct gct gat 1591

Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala Asp

185 190 195 aaa gct gcc tgc ctg ttg cca aag ctc gat gaa ctt egg gat gaa ggg 1639

Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly

200 205 210 aag gct tcg tct gcc aaa cag aga ctc aag tgt gcc agt ctc caa aaa 1687

Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys Ala Ser Leu Gin Lys

215 220 225 ttt gga gaa aga gct ttc aaa gca tgg gca gta gct cgc ctg agc cag 1735

Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gin

230 235 240 245 aga ttt ccc aaa gct gag ttt gca gaa gtt tcc aag tta gtg aca gat 1783

Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp

250 255 260 ctt acc aaa gtc cac acg gaa tgc tgc cat gga gat ctg ctt gaa tgt 1831

Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys

265 270 275 gct gat gac agg gcg gac ctt gcc aag tat atc tgt gaa aat caa gat 1879

Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gin Asp

280 285 290 tcg atc tcc agt aaa ctg aag gaa tgc tgt gaa aaa cct ctg ttg gaa 1927

Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu

295 300 305 aaa tcc cac tgc att gcc gaa gtg gaa aat gat gag atg cct gct gac 1975

Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp

310 315 320 325 ttg cct tca tta gct gct gat ttt gtt gaa agt aag gat gtt tgc aaa 2023

Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys

330 335 340 aac tat gct gag gca aag gat gtc ttc ctg ggc atg ttt ttg tat gaa 2071

Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu

345 350 355 tat gca aga agg cat cct gat tac tct gtc gtg ctg ctg ctg aga ctt 2119

Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu

360 365 370 gcc aag aca tat gaa acc act cta gag aag tgc tgt gcc gct gca gat 2167

Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp

375 380 385 cct cat gaa tgc tat gcc aaa gtg ttc gat gaa ttt aaa cct ctt gtg 2215 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 390 395 400 405 gaa gag cct cag aat tta atc aaa caa aat tgt gag ctt ttt gag cag 2263

Glu Glu Pro Gin Asn Leu Ile Lys Gin Asn Cys Glu Leu Phe Glu Gin

410 415 420 ctt gga gag tac aaa ttc cag aat gcg cta tta gtt cgt tac acc aag 2311

Leu Gly Glu Tyr Lys Phe Gin Asn Ala Leu Leu Val Arg Tyr Thr Lys

425 430 435 aaa gta ccc caa gtg tca act cca act ctt gta gag gtc tca aga aac 2359

Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn

440 445 450 cta gga aaa gtg ggc agc aaa tgt tgt aaa cat cct gaa gca aaa aga 2407

Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg

455 460 465 atg ccc tgt gca gaa gac tat cta tcc gtg gtc ctg aac cag tta tgt 2455

Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gin Leu Cys

470 475 480 485 gtg ttg cat gag aaa acg cca gta agt gac aga gtc acc aaa tgc tgc 2503 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys

490 495 500 aca gaa tcc ttg gtg aac agg cga cca tgc ttt tca gct ctg gaa gtc 2551

Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val

505 510 515 gat gaa aca tac gtt ccc aaa gag ttt aat gct gaa aca ttc acc ttc 2599

Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe

520 525 530 cat gca gat ata tgc aca ctt tct gag aag gag aga caa atc aag aaa 2647

His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gin Ile Lys Lys

535 540 545 caa act gca ctt gtt gag ctc gtg aaa cac aag ccc aag gca aca aaa 2695

Gin Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys

550 555 560 565 gag caa ctg aaa gct gtt atg gat gat ttc gca gct ttt gta gag aag 2743

Glu Gin Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys

570 575 580 tgc tgc aag gct gac gat aag gag acc tgc ttt gcc gag gag ggt aaa 2791

Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys

585 590 595 aaa ctt gtt gct gca agt caa gct gcc tta ggc tta taa 2830

Lys Leu Val Ala Ala Ser Gin Ala Ala Leu Gly Leu

600 605

<210> 156

<211> 609

<212> PRT

<213> synthetic <400> 156

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110

Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe Leu Gin 115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 135 140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190

Cys Gin Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gin Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gin Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240

Ala Arg Leu Ser Gin Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285

Cys Glu Asn Gin Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 375 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gin Asn Leu Ile Lys Gin Asn Cys

405 410 415

Glu Leu Phe Glu Gin Leu Gly Glu Tyr Lys Phe Gin Asn Ala Leu Leu

420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 455 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480

Leu Asn Gin Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

530 535 540

Arg Gin Ile Lys Lys Gin Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560

Pro Lys Ala Thr Lys Glu Gin Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gin Ala Ala Leu Gly 595 600 605

Leu

<210> 157

<211> 3505

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (3505)

<223> construct KU70 (up) -pPDIl-kanMX-terminator-KU70

<220>

<221> misc_feature

<222> (1) . . (1002 )

<223> upstream region

<220>

<221> promoter

<222> (1002 ) . . (1542 )

<223> promoter pPDIl

<220>

<221> CDS

<222> (1543) . . (2352 )

<223> kanMX

<220>

<221> terminator

<222> (2353) . . (2555)

<223> terminator <220>

<221> misc_feature

<222> (2556) . . (3505)

<223> downstream region

<400> 157 gctcttcaga agcctaactt taatggggta aaatatcagc agtaggctga atatagagca 60 tggcaacgtt tacgtcaaca gagtttaata gtgaaaacta cgataggtte aggcctgtat 120 acccagacga gttgtatcag caattagtgg attatcatgt tggtgctaaa ggattgtgtg 180 tcgatgttgg atgtggctct ggccaggcaa cgttcaccct gaaaaagtac tttgacaagg 240 tcataggatc tgatatctca gaaaatcagt tggcagtggc taggaaaaga cagcctgctg 300 gaattgaatt taggttagga aegggcgagg atttttcttg gctaaeggag acgccagatg 360 taatcaccgc egctgaatge ctgcactggg tagacccaca gaaattcgtt gcaaatgtcg 420 ccaattctct aagggatcat ggcaccttga gttactggct ttatacagaa ccaatttttc 480 agaacgaaag ggccaatcag gtttacaaca aattcaccta cgggtctgac tacctgggcc 540 catattggga tcctggccgt acacatttca gaaatcattt aaaggagttg aatcacatct 600 taetggatag cgagcttttt gacgaagtga aaatttctaa ttttaaacaa gaggaagggg 660 tcaaaaaegg agatatctta tacttggaaa aagagatgac aatcagtgat ttcatcaatt 720 ttgtatctag ttggccttct gtgttttcgt ggaagcagca acgaggaaag gagggtatcc 780 tagatgattt ttacaacgaa ctgaacgact gctttgaggg gggtaacatg aaagtaatat 840 ggaactccgt cctagtattt gccaggagga agcaaagggt tgtataggct ttagtactta 900 tagaggaaac ggggttacgt gcaagcgcgc atgcctgagc tttgaggggg gggactttca 960 catctcttct tctcacactt agccctaaca cagagatcaa ctactcaagg cagcaccaaa 1020 agacgccaga aattatatgg tccaggaaaa aacaactctc tttactacaa aatttgaaca 1080 ggtgtcctgc ctgcggaaac tacaaacgat cacacaccct etgeatgeat tgcgtaggac 1140 aaatcaggag acattggaac gactctgttc ctcaacagga ggcatttcgt gaagagtttg 1200 ttaatccttt ggatgagaag attctttatc caggaaagaa agaactgccc gatgaacgaa 1260 ctttaegtaa gaaggagtgg etgaagagaa gaccccgaac actccctgtt gaatagaaca 1320 cgaacactgt aaatagaata aaagaaaact tggatagtag aacttcaatg tagtgtttct 1380 attgtcttac gcggctcttt agattgeaat ccccagaatg gaatcgtcca tctttctcaa 1440 cccactcaaa gataatctac cagacatacc tacgccctcc atcccagcac cacgtcgcga 1500 tcacccctaa aacttcaata attgaacacg taetgattte ca atg ggt aag gaa 1554 Met Gly Lys Glu 1 aag act cac gtt tcg agg ccg cga tta aat tcc aac atg gat gct gat 1602 Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn Met Asp Ala Asp 5 10 15 20 tta tat ggg tat aaa tgg gct cgc gat aat gtc ggg caa tca ggt gcg 1650 Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly Gin Ser Gly Ala 25 30 35 aca atc tat cga ttg tat ggg aag ccc gat gcg cca gag ttg ttt ctg 1698

Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro Glu Leu Phe Leu

40 45 50 aaa cat ggc aaa ggt agc gtt gcc aat gat gtt aca gat gag atg gtc 1746

Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr Asp Glu Met Val

55 60 65 aga cta aac tgg ctg acg gaa ttt atg cct ctt ccg acc atc aag cat 1794

Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro Thr Ile Lys His

70 75 80 ttt atc cgt act cct gat gat gca tgg tta ctc acc act gcg atc ccc 1842

Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr Thr Ala Ile Pro

85 90 95 100 ggc aaa aca gca ttc cag gta tta gaa gaa tat cct gat tca ggt gaa 1890

Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro Asp Ser Gly Glu

105 110 115 aat att gtt gat gcg ctg gca gtg ttc ctg cgc egg ttg cat tcg att 1938

Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg Leu His Ser Ile

120 125 130 cct gtt tgt aat tgt cct ttt aac agc gat cgc gta ttt cgt ctc gct 1986

Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val Phe Arg Leu Ala

135 140 145 cag gcg caa tca cga atg aat aac ggt ttg gtt gat gcg agt gat ttt 2034

Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp Ala Ser Asp Phe

150 155 160 gat gac gag cgt aat ggc tgg cct gtt gaa caa gtc tgg aaa gaa atg 2082

Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val Trp Lys Glu Met

165 170 175 180 cat aag ctt ttg cca ttc tca ccg gat tca gtc gtc act cat ggt gat 2130

His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val Thr His Gly Asp

185 190 195 ttc tca ctt gat aac ctt att ttt gac gag ggg aaa tta ata ggt tgt 2178

Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys Leu Ile Gly Cys

200 205 210 att gat gtt gga cga gtc gga atc gca gac cga tac cag gat ctt gcc 2226 Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr Gin Asp Leu Ala

215 220 225 atc cta tgg aac tgc ctc ggt gag ttt tct cct tca tta cag aaa egg 2274 Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser Leu Gin Lys Arg

230 235 240 ctt ttt caa aaa tat ggt att gat aat cct gat atg aat aaa ttg cag 2322

Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met Asn Lys Leu Gin

245 250 255 260 ttt cat ttg atg ctc gat gag ttt ttc taa tcagtaetga caataaaaag 2372

Phe His Leu Met Leu Asp Glu Phe Phe 265 attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt 2432 taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg 2492 aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata 2552 ctgttcatct tagagaatgt cagtgaggtt atttctgagc taatcattac cttgcccggt 2612 acaggaatag ggtgttacct tattaattac gacggtggtc aaaacgacga aatttacccc 2672 atttttgagt tacaagacct gaatttggaa atgatgaaac aattgtacca agtcttggag 2732 gaccatgtaa gtgggcttaa tcctctcgag aagcaattcc caattgaaca cagtaaaccg 2792 ttatcagcca ctctgttctt tcacttaagg tctctttttt acatggcgaa gactcataag 2852 cgtactggaa gacattacaa cttgaaaaag attttcttgt tcactaataa cgataaacct 2912 tacaatggaa actctcagct gagagttccc ttgaagaaaa ccctggctga ttacaatgac 2972 gtagacatta ctttgattcc gtttcttctg aacaagcctt caggtgtcaa gtttgacaag 3032 acggaatact cagaaatttt gttctatgat aaagatgctt gttcgatgtc aattgaggag 3092 atccgccaac gaatttctag acataaggag atcaagcggg tttacttcac ctgtcctttg 3152 aaaatcgcaa ataacttgtg catttctgtg aaaggttatt ctatgtttta tcatgaaact 3212 ccaaggaaga tcaaatttgt cgtcaatgag ggttcaactt tcaaagatgt ggagacaaaa 3272 tctcagtttg tcgatccaac atccggaaaa gagttttcca gtgaacagct gatcaaagca 3332 tatcctctag gtgccgatgc ttacattcct ttaaactcag agcaagtcaa aacaataaat 3392 cgatttaatg atatcatcaa tatcccctct ttggaaattc taggtttcag ggatatatct 3452 aattggttgc cacagtatca gtttggcaaa gcatcgtttt tatcccctaa taa 3505

<210> 158

<211> 269

<212> PRT

<213> synthetic

<400> 158

Met Gly Lys Glu Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn 1 5 10 15

Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly 20 25 30

Gin Ser Gly Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro 35 40 45

Glu Leu Phe Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr 50 55 60 Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro 65 70 75 80

Thr Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr 85 90 95

Thr Ala Ile Pro Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro 100 105 110

Asp Ser Gly Glu Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg 115 120 125

Leu His Ser Ile Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val 130 135 140

Phe Arg Leu Ala Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp 145 150 155 160

Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val 165 170 175

Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val 180 185 190

Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys 195 200 205

Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr

210 215 220

Gin Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser 225 230 235 240

Leu Gin Lys Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met 245 250 255

Asn Lys Leu Gin Phe His Leu Met Leu Asp Glu Phe Phe 260 265

<210> 159

<211> 3533

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (3533)

<223> KU70 (up) -pFPR2-kanMX-terminator-KU70 construct <220>

<221> misc_feature

<222> (1) . . (1002 )

<223> KU70 upstream region

<220>

<221> promoter

<222> (1003) . . (1570)

<223> promoter pFPR2

<220>

<221> CDS

<222> (1571) . . (2380)

<223> kanMX

<220>

<221> terminator

<222> (2381) . . (2583)

<223> terminator

<220>

<221> misc_feature

<222> (2584 ) . . (3533)

<223> KU70 downstream region

<400> 159 gctcttcaga agcctaactt taatggggta aaatatcagc agtaggctga atatagagca 60 tggcaacgtt tacgtcaaca gagtttaata gtgaaaacta cgataggttc aggcctgtat 120 acccagacga gttgtatcag caattagtgg attatcatgt tggtgctaaa ggattgtgtg 180 tcgatgttgg atgtggctct ggccaggcaa cgttcaccct gaaaaagtac tttgacaagg 240 tcataggatc tgatatctca gaaaatcagt tggcagtggc taggaaaaga cagcctgctg 300 gaattgaatt taggttagga acgggcgagg atttttcttg gctaacggag acgccagatg 360 taatcaccgc cgctgaatgc ctgcactggg tagacccaca gaaattcgtt gcaaatgtcg 420 ccaattctct aagggatcat ggcaccttga gttactggct ttatacagaa ccaatttttc 480 agaacgaaag ggccaatcag gtttacaaca aattcaccta cgggtctgac tacctgggcc 540 catattggga tcctggccgt acacatttca gaaatcattt aaaggagttg aatcacatct 600 tactggatag cgagcttttt gacgaagtga aaatttctaa ttttaaacaa gaggaagggg 660 tcaaaaacgg agatatctta tacttggaaa aagagatgac aatcagtgat ttcatcaatt 720 ttgtatctag ttggccttct gtgttttcgt ggaagcagca acgaggaaag gagggtatcc 780 tagatgattt ttacaacgaa ctgaacgact gctttgaggg gggtaacatg aaagtaatat 840 ggaactccgt cctagtattt gccaggagga agcaaagggt tgtataggct ttagtactta 900 tagaggaaac ggggttacgt gcaagcgcgc atgcctgagc tttgaggggg gggactttca 960 catctcttct tctcacactt agccctaaca cagagatcaa ctacaagcgg caattcctac 1020 agaaagtgtt tgcaaagttc ctaatctcgg tttgtcacca agtcactttt ctacgaaccc 1080 tgagtttcct cacgaggaat caagttttga aatgcccgat ttgtcgcagt ttgatagttt 1140 tgatgaattt tttaatggaa tctttccaga atttaaaaga gtcaagaata tttaacttgt 1200 gagcgaacga acatgaaaac cgaaagaata gttgatgatg atgttgaagc aatgcaagta 1260 ccatataaat taggagaagt cctttgtgta ttggcgattc cttttgatgc aagtgagcgt 1320 aaatggttaa caggctgata catttactca agctagtagt gaaagccatt gcgaaatcag 1380 tttatcatag tagtggacgt cacaatggaa ctcatctctg cgaatcgttg tagaataact 1440 gtaggtaaat atgcaattct aacttctggt ctaaaatgtt cagtgacaat gcttatttct 1500 gtagtcgcga agaattgctt ctgaggagac ggagcgagcc atgtattctc gccaacttga 1560 caagaacgcc atg ggt aag gaa aag act cac gtt tcg agg ccg cga tta 1609

Met Gly Lys Glu Lys Thr His Val Ser Arg Pro Arg Leu

1 5 10 aat tcc aac atg gat gct gat tta tat ggg tat aaa tgg gct cgc gat 1657

Asn Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp

15 20 25 aat gtc ggg caa tca ggt gcg aca atc tat cga ttg tat ggg aag ccc 1705

Asn Val Gly Gin Ser Gly Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro

30 35 40 45 gat gcg cca gag ttg ttt ctg aaa cat ggc aaa ggt agc gtt gcc aat 1753

Asp Ala Pro Glu Leu Phe Leu Lys His Gly Lys Gly Ser Val Ala Asn

50 55 60 gat gtt aca gat gag atg gtc aga cta aac tgg ctg acg gaa ttt atg 1801

Asp Val Thr Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met

65 70 75 cct ctt ccg acc atc aag cat ttt atc cgt act cct gat gat gca tgg 1849

Pro Leu Pro Thr Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp

80 85 90 tta ctc acc act gcg atc ccc ggc aaa aca gca ttc cag gta tta gaa 1897

Leu Leu Thr Thr Ala Ile Pro Gly Lys Thr Ala Phe Gin Val Leu Glu

95 100 105 gaa tat cct gat tca ggt gaa aat att gtt gat gcg ctg gca gtg ttc 1945

Glu Tyr Pro Asp Ser Gly Glu Asn Ile Val Asp Ala Leu Ala Val Phe

110 115 120 125 ctg cgc egg ttg cat tcg att cct gtt tgt aat tgt cct ttt aac agc 1993

Leu Arg Arg Leu His Ser Ile Pro Val Cys Asn Cys Pro Phe Asn Ser

130 135 140 gat cgc gta ttt cgt ctc gct cag gcg caa tca cga atg aat aac ggt 2041

Asp Arg Val Phe Arg Leu Ala Gin Ala Gin Ser Arg Met Asn Asn Gly

145 150 155 ttg gtt gat gcg agt gat ttt gat gac gag cgt aat ggc tgg cct gtt 2089

Leu Val Asp Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val

160 165 170 gaa caa gtc tgg aaa gaa atg cat aag ctt ttg cca ttc tca ccg gat 2137

Glu Gin Val Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp

175 180 185 tca gtc gtc act cat ggt gat ttc tca ctt gat aac ctt att ttt gac 2185 Ser Val Val Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp 190 195 200 205 gag ggg aaa tta ata ggt tgt att gat gtt gga cga gtc gga atc gca 2233 Glu Gly Lys Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala 210 215 220 gac cga tac cag gat ctt gcc atc cta tgg aac tgc ctc ggt gag ttt 2281 Asp Arg Tyr Gin Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe 225 230 235 tct cct tca tta cag aaa egg ctt ttt caa aaa tat ggt att gat aat 2329 Ser Pro Ser Leu Gin Lys Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn 240 245 250 cct gat atg aat aaa ttg cag ttt cat ttg atg ctc gat gag ttt ttc 2377 Pro Asp Met Asn Lys Leu Gin Phe His Leu Met Leu Asp Glu Phe Phe 255 260 265 taa tcagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta 2430 tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt 2490 tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc 2550 gtcaatcgta tgtgaatgct ggtcgctata ctgttcatct tagagaatgt cagtgaggtt 2610 atttctgagc taatcattac cttgcccggt acaggaatag ggtgttacct tattaattac 2670 gacggtggtc aaaacgacga aatttacccc atttttgagt tacaagacct gaatttggaa 2730 atgatgaaac aattgtacca agtcttggag gaccatgtaa gtgggcttaa tcctctcgag 2790 aagcaattcc caattgaaca cagtaaaccg ttatcagcca ctctgttctt tcacttaagg 2850 tctctttttt acatggcgaa gactcataag cgtactggaa gacattacaa cttgaaaaag 2910 attttcttgt tcactaataa cgataaacct tacaatggaa actctcagct gagagttccc 2970 ttgaagaaaa ccctggctga ttacaatgac gtagacatta ctttgattcc gtttcttctg 3030 aacaagcctt caggtgtcaa gtttgacaag acggaatact cagaaatttt gttctatgat 3090 aaagatgctt gttcgatgtc aattgaggag atccgccaac gaatttctag acataaggag 3150 atcaagcggg tttacttcac ctgtcctttg aaaatcgcaa ataacttgtg catttctgtg 3210 aaaggttatt ctatgtttta tcatgaaact ccaaggaaga tcaaatttgt cgtcaatgag 3270 ggttcaactt tcaaagatgt ggagacaaaa tctcagtttg tcgatccaac atccggaaaa 3330 gagttttcca gtgaacagct gatcaaagca tatcctctag gtgccgatgc ttacattcct 3390 ttaaactcag agcaagtcaa aacaataaat cgatttaatg atatcatcaa tatcccctct 3450 ttggaaattc taggtttcag ggatatatct aattggttgc cacagtatca gtttggcaaa 3510 gcatcgtttt tatcccctaa taa 3533

<210> 160 <211> 269

<212> PRT

<213> synthetic

<400> 160

Met Gly Lys Glu Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn

1 5 10 15

Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly 20 25 30

Gin Ser Gly Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro 35 40 45

Glu Leu Phe Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr 50 55 60

Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro 65 70 75 80

Thr Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr 85 90 95

Thr Ala Ile Pro Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro 100 105 110

Asp Ser Gly Glu Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg 115 120 125

Leu His Ser Ile Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val 130 135 140

Phe Arg Leu Ala Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp 145 150 155 160

Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val 165 170 175

Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val 180 185 190

Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys 195 200 205

Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr 210 215 220

Gin Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser 225 230 235 240

Leu Gin Lys Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met

245 250 255

Asn Lys Leu Gin Phe His Leu Met Leu Asp Glu Phe Phe

260 265

<210> 161

<211> 3632

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (3632 )

<223> construct KU70 (up) -pRPL3-kanMX-terminator-KU70 (dw)

<220>

<221> misc_feature

<222> (1) . . (1002 )

<223> KU70 upstream region

<220>

<221> promoter

<222> (1003) . . (1669)

<223> pRPL3 promoter

<220>

<221> CDS

<222> (1670) . . (2479)

<223> kanMX

<220>

<221> terminator

<222> (2480) . . (2682 )

<223> terminator

<220>

<221> misc_feature

<222> (2480) . . (2682 )

<223> KU70 downstream region

<400> 161 gctcttcaga agcctaactt taatggggta aaatatcagc agtaggctga atatagagca 60 tggcaacgtt tacgtcaaca gagtttaata gtgaaaacta cgataggttc aggcctgtat 120 acccagacga gttgtatcag caattagtgg attatcatgt tggtgctaaa ggattgtgtg 180 tcgatgttgg atgtggctct ggccaggcaa cgttcaccct gaaaaagtac tttgacaagg 240 tcataggatc tgatatctca gaaaatcagt tggcagtggc taggaaaaga cagcctgctg 300 gaattgaatt taggttagga acgggcgagg atttttcttg gctaacggag acgccagatg 360 taatcaccgc cgctgaatgc ctgcactggg tagacccaca gaaattcgtt gcaaatgtcg 420 ccaattctct aagggatcat ggcaccttga gttactggct ttatacagaa ccaatttttc 480 agaacgaaag ggccaatcag gtttacaaca aattcaccta cgggtctgac tacctgggcc 540 catattggga tcctggccgt acacatttca gaaatcattt aaaggagttg aatcacatct 600 taetggatag cgagcttttt gacgaagtga aaatttctaa ttttaaacaa gaggaagggg 660 tcaaaaaegg agatatctta tacttggaaa aagagatgac aatcagtgat ttcatcaatt 720 ttgtatctag ttggccttct gtgttttcgt ggaagcagca acgaggaaag gagggtatcc 780 tagatgattt ttacaacgaa ctgaacgact gctttgaggg gggtaacatg aaagtaatat 840 ggaactccgt cctagtattt gccaggagga agcaaagggt tgtataggct ttagtactta 900 tagaggaaac ggggttacgt gcaagcgcgc atgcctgagc tttgaggggg gggactttca 960 catctcttct tctcacactt agccctaaca cagagatcaa ctccaacgac ttgaggaaat 1020 gacgattatt tcttaagata ttcatggtga ggttttccaa atgtgatctg aaatgtaatc 1080 ttggcaagtt tggttcataa aacgtcaaaa gaccacctga agataaaaaa taaagtggaa 1140 ggagaagaaa aagaggtttt tttccagatg agtctatgac taagatttct cttcaaagaa 1200 ttttttcaag gtttgagagg gatgaaagcg aggctgctge acgattatcg atgtcattca 1260 atagggaggt agaagagaac tgagggagaa aaggctacaa ctgaacctaa aactaactga 1320 atttggaatg aagcctatta cacgagaggt gtggcgcctt tccaaagtca tcaggttcaa 1380 gttattgctt tctggagcaa taaatgatgt caatcccata cggggtctcc acccttatag 1440 aattacttat ctaccggcta ggtgttgcat cgcggaccag cccaatcggg taattttagt 1500 tgaaaaggtt tttagcgcga gtctagttat tacccggata gtctaatgct gaaaatttct 1560 cggacatttt tgatccgtac gtatatttca gtacggagaa tcatcaacaa gaatctttca 1620 atgccaccaa aaaaaaagac atttttcacc atcgcgcatt cacagtagc atg ggt aag 1678 Met Gly Lys 1 gaa aag act cac gtt tcg agg ccg cga tta aat tcc aac atg gat gct 1726 Glu Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn Met Asp Ala 5 10 15 gat tta tat ggg tat aaa tgg gct cgc gat aat gtc ggg caa tca ggt 1774 Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly Gin Ser Gly 20 25 30 35 gcg aca atc tat cga ttg tat ggg aag ccc gat gcg cca gag ttg ttt 1822 Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro Glu Leu Phe 40 45 50 ctg aaa cat ggc aaa ggt agc gtt gcc aat gat gtt aca gat gag atg 1870 Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr Asp Glu Met 55 60 65 gtc aga cta aac tgg ctg acg gaa ttt atg cct ctt ccg acc atc aag 1918 Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro Thr Ile Lys 70 75 80 cat ttt atc cgt act cct gat gat gca tgg tta ctc acc act gcg atc 1966 His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr Thr Ala Ile 85 90 95 ccc ggc aaa aca gca ttc cag gta tta gaa gaa tat cct gat tca ggt 2014 Pro Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro Asp Ser Gly 100 105 110 115 gaa aat att gtt gat gcg ctg gca gtg ttc ctg cgc cgg ttg cat tcg 2062 Glu Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg Leu His Ser 120 125 130 att cct gtt tgt aat tgt cct ttt aac agc gat cgc gta ttt cgt ctc 2110 Ile Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val Phe Arg Leu 135 140 145 gct cag gcg caa tca cga atg aat aac ggt ttg gtt gat gcg agt gat 2158 Ala Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp Ala Ser Asp 150 155 160 ttt gat gac gag cgt aat ggc tgg cct gtt gaa caa gtc tgg aaa gaa 2206 Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val Trp Lys Glu 165 170 175 atg cat aag ctt ttg cca ttc tca ccg gat tca gtc gtc act cat ggt 2254 Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val Thr His Gly 180 185 190 195 gat ttc tca ctt gat aac ctt att ttt gac gag ggg aaa tta ata ggt 2302 Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys Leu Ile Gly 200 205 210 tgt att gat gtt gga cga gtc gga atc gca gac cga tac cag gat ctt 2350 Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr Gin Asp Leu 215 220 225 gcc atc cta tgg aac tgc ctc ggt gag ttt tct cct tca tta cag aaa 2398 Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser Leu Gin Lys 230 235 240 egg ctt ttt caa aaa tat ggt att gat aat cct gat atg aat aaa ttg 2446 Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met Asn Lys Leu 245 250 255 cag ttt cat ttg atg ctc gat gag ttt ttc taa tcagtaetga caataaaaag 2499 Gin Phe His Leu Met Leu Asp Glu Phe Phe 260 265 attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt 2559 taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg 2619 aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata 2679 ctgttcatct tagagaatgt cagtgaggtt atttctgagc taatcattac cttgcccggt 2739 acaggaatag ggtgttacct tattaattac gaeggtggte aaaacgacga aatttacccc 2799 atttttgagt tacaagacct gaatttggaa atgatgaaac aattgtacca agtcttggag 2859 gaccatgtaa gtgggcttaa tcctctcgag aagcaattcc caattgaaca cagtaaaccg 2919 ttatcagcca ctctgttctt tcacttaagg tctctttttt acatggcgaa gactcataag 2979 cgtactggaa gacattacaa cttgaaaaag attttcttgt tcactaataa cgataaacct 3039 tacaatggaa actctcagct gagagttccc ttgaagaaaa ccctggctga ttacaatgac 3099 gtagacatta ctttgattcc gtttcttctg aacaagcctt caggtgtcaa gtttgacaag 3159 acggaatact cagaaatttt gttctatgat aaagatgctt gttcgatgtc aattgaggag 3219 atccgccaac gaatttctag acataaggag atcaagcggg tttacttcac ctgtcctttg 3279 aaaatcgcaa ataacttgtg catttctgtg aaaggttatt ctatgtttta tcatgaaact 3339 ccaaggaaga tcaaatttgt cgtcaatgag ggttcaactt tcaaagatgt ggagacaaaa 3399 tctcagtttg tcgatccaac atccggaaaa gagttttcca gtgaacagct gatcaaagca 3459 tatcctctag gtgccgatgc ttacattcct ttaaactcag agcaagtcaa aacaataaat 3519 cgatttaatg atatcatcaa tatcccctct ttggaaattc taggtttcag ggatatatct 3579 aattggttgc cacagtatca gtttggcaaa gcatcgtttt tatcccctaa taa 3632

<210> 162

<211> 269

<212> PRT

<213> synthetic

<400> 162

Met Gly Lys Glu Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn 1 5 10 15

Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly 20 25 30

Gin Ser Gly Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro 35 40 45

Glu Leu Phe Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr 50 55 60

Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro 65 70 75 80

Thr Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr 85 90 95

Thr Ala Ile Pro Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro 100 105 110

Asp Ser Gly Glu Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg 115 120 125 Leu His Ser Ile Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val

130 135 140

Phe Arg Leu Ala Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp 145 150 155 160

Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val

165 170 175

Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val 180 185 190

Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys 195 200 205

Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr

210 215 220

Gin Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser 225 230 235 240

Leu Gin Lys Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met

245 250 255

Asn Lys Leu Gin Phe His Leu Met Leu Asp Glu Phe Phe 260 265

<210> 163

<211> 3573

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (3573)

<223> construct KU70 (up) -pRPL6A-kanMX-terminator-KU70 (dw)

<220>

<221> misc_feature

<222> (1) . . (1002 )

<223> KU70 upstream region

<220>

<221> promoter

<222> (1003) . . (1610)

<223> promoter pRPL6A

<220>

<221> CDS

<222> (1611) . . (2420)

<223> kanMX

<220> <221> terminator

<222> (2421) . . (2623)

<223> terminator

<220>

<221> misc_feature

<222> (2624 ) . . (3573)

<223> KU70 downstream region

<400> 163 gctcttcaga agcctaactt taatggggta aaatatcagc agtaggctga atatagagca 60 tggcaacgtt tacgtcaaca gagtttaata gtgaaaacta cgataggttc aggcctgtat 120 acccagacga gttgtatcag caattagtgg attatcatgt tggtgctaaa ggattgtgtg 180 tcgatgttgg atgtggctct ggccaggcaa cgttcaccct gaaaaagtac tttgacaagg 240 tcataggatc tgatatctca gaaaatcagt tggcagtggc taggaaaaga cagcctgctg 300 gaattgaatt taggttagga acgggcgagg atttttcttg gctaacggag acgccagatg 360 taatcaccgc cgctgaatgc ctgcactggg tagacccaca gaaattcgtt gcaaatgtcg 420 ccaattctct aagggatcat ggcaccttga gttactggct ttatacagaa ccaatttttc 480 agaacgaaag ggccaatcag gtttacaaca aattcaccta cgggtctgac tacctgggcc 540 catattggga tcctggccgt acacatttca gaaatcattt aaaggagttg aatcacatct 600 tactggatag cgagcttttt gacgaagtga aaatttctaa ttttaaacaa gaggaagggg 660 tcaaaaacgg agatatctta tacttggaaa aagagatgac aatcagtgat ttcatcaatt 720 ttgtatctag ttggccttct gtgttttcgt ggaagcagca acgaggaaag gagggtatcc 780 tagatgattt ttacaacgaa ctgaacgact gctttgaggg gggtaacatg aaagtaatat 840 ggaactccgt cctagtattt gccaggagga agcaaagggt tgtataggct ttagtactta 900 tagaggaaac ggggttacgt gcaagcgcgc atgcctgagc tttgaggggg gggactttca 960 catctcttct tctcacactt agccctaaca cagagatcaa ctcgcttcag catttcgaca 1020 gtgtaaagaa gctcatctca cttgaccaga atgcttttgc tatctgttat ttcaaagacc 1080 tagagtacct cccaattgcc atagaagctt ggaaacttta tgaaccttct tctaaagtac 1140 cggtggtatt cgctcaagtg acacaactcc ccaaatcagc tccaatcgaa tggagcgggt 1200 cagtgtatca agagacacca aactacgatg attactacgg tgattcgtca gaaaatgaag 1260 atgagtccac acagattgcc aacaacttgt cctcgctaga attacaggga caatcggtca 1320 catttcaaga cggctacgtt agcttgacat tcgccccagg cctatcagcc acagcacaca 1380 acacggtaat tttaaacaag gggcaggcaa gcaagctgga aaccattcca caggaatatt 1440 ccgtcgacat catccccgtc aacagactgt tcgatttcac agggaaagat caggacctag 1500 ccgtactcca caggctgaac atctgagatc aggcatgaga atccttacac catctatgtt 1560 gctctagggc tgtcaaccct attatgcacg tgacggtgcg actctttcaa atg ggt 1616 Met Gly 1 aag gaa aag act cac gtt tcg agg ccg cga tta aat tcc aac atg gat 1664

Lys Glu Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn Met Asp

5 10 15 gct gat tta tat ggg tat aaa tgg gct cgc gat aat gtc ggg caa tca 1712

Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly Gin Ser

20 25 30 ggt gcg aca atc tat cga ttg tat ggg aag ccc gat gcg cca gag ttg 1760

Gly Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro Glu Leu

35 40 45 50 ttt ctg aaa cat ggc aaa ggt agc gtt gcc aat gat gtt aca gat gag 1808

Phe Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr Asp Glu

55 60 65 atg gtc aga cta aac tgg ctg acg gaa ttt atg cct ctt ccg acc atc 1856

Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro Thr Ile

70 75 80 aag cat ttt atc cgt act cct gat gat gca tgg tta ctc acc act gcg 1904

Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr Thr Ala

85 90 95 atc ccc ggc aaa aca gca ttc cag gta tta gaa gaa tat cct gat tca 1952 Ile Pro Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro Asp Ser

100 105 110 ggt gaa aat att gtt gat gcg ctg gca gtg ttc ctg cgc egg ttg cat 2000

Gly Glu Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg Leu His

115 120 125 130 tcg att cct gtt tgt aat tgt cct ttt aac agc gat cgc gta ttt cgt 2048

Ser Ile Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val Phe Arg

135 140 145 ctc gct cag gcg caa tca cga atg aat aac ggt ttg gtt gat gcg agt 2096

Leu Ala Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp Ala Ser

150 155 160 gat ttt gat gac gag cgt aat ggc tgg cct gtt gaa caa gtc tgg aaa 2144

Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val Trp Lys

165 170 175 gaa atg cat aag ctt ttg cca ttc tca ccg gat tca gtc gtc act cat 2192

Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val Thr His

180 185 190 ggt gat ttc tca ctt gat aac ctt att ttt gac gag ggg aaa tta ata 2240

Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys Leu Ile

195 200 205 210 ggt tgt att gat gtt gga cga gtc gga atc gca gac cga tac cag gat 2288

Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr Gin Asp

215 220 225 ctt gcc atc cta tgg aac tgc ctc ggt gag ttt tct cct tca tta cag 2336

Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser Leu Gin

230 235 240 aaa egg ctt ttt caa aaa tat ggt att gat aat cct gat atg aat aaa 2384 Lys Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met Asn Lys 245 250 255 ttg cag ttt cat ttg atg ctc gat gag ttt ttc taa tcagtaetga 2430 Leu Gin Phe His Leu Met Leu Asp Glu Phe Phe

260 265 caataaaaag attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt 2490 tgttctattt taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg 2550 cccagatgcg aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct 2610 ggtcgctata ctgttcatct tagagaatgt cagtgaggtt atttctgagc taatcattac 2670 cttgcccggt acaggaatag ggtgttacct tattaattac gaeggtggte aaaacgacga 2730 aatttacccc atttttgagt tacaagacct gaatttggaa atgatgaaac aattgtacca 2790 agtcttggag gaccatgtaa gtgggcttaa tcctctcgag aagcaattcc caattgaaca 2850 cagtaaaccg ttatcagcca ctctgttctt tcacttaagg tctctttttt acatggcgaa 2910 gactcataag cgtactggaa gacattacaa cttgaaaaag attttcttgt tcactaataa 2970 cgataaacct tacaatggaa actctcagct gagagttccc ttgaagaaaa ccctggctga 3030 ttacaatgac gtagacatta ctttgattcc gtttcttctg aacaagcctt caggtgtcaa 3090 gtttgacaag acggaatact cagaaatttt gttctatgat aaagatgctt gttcgatgtc 3150 aattgaggag atccgccaac gaatttctag acataaggag atcaagcggg tttacttcac 3210 ctgtcctttg aaaatcgcaa ataacttgtg catttctgtg aaaggttatt ctatgtttta 3270 tcatgaaact ccaaggaaga tcaaatttgt egtcaatgag ggttcaactt tcaaagatgt 3330 ggagacaaaa tctcagtttg tcgatccaac atccggaaaa gagttttcca gtgaacagct 3390 gatcaaagca tatcctctag gtgccgatgc ttacattcct ttaaactcag agcaagtcaa 3450 aacaataaat cgatttaatg atatcatcaa tatcccctct ttggaaattc taggtttcag 3510 ggatatatct aattggttgc cacagtatca gtttggcaaa gcatcgtttt tatcccctaa 3570 taa 3573

<210> 164

<211> 269

<212> PRT

<213> synthetic

<400> 164

Met Gly Lys Glu Lys Thr His Val Ser Arg Pro Arg Leu Asn Ser Asn 1 5 10 15

Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn Val Gly 20 25 30 Gin Ser Gly Ala Thr Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro 35 40 45

Glu Leu Phe Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr 50 55 60

Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro 65 70 75 80

Thr Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu Thr 85 90 95

Thr Ala Ile Pro Gly Lys Thr Ala Phe Gin Val Leu Glu Glu Tyr Pro 100 105 110

Asp Ser Gly Glu Asn Ile Val Asp Ala Leu Ala Val Phe Leu Arg Arg 115 120 125

Leu His Ser Ile Pro Val Cys Asn Cys Pro Phe Asn Ser Asp Arg Val 130 135 140

Phe Arg Leu Ala Gin Ala Gin Ser Arg Met Asn Asn Gly Leu Val Asp 145 150 155 160

Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gin Val

165 170 175

Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val 180 185 190

Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys 195 200 205

Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg Tyr 210 215 220

Gin Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe Ser Pro Ser 225 230 235 240

Leu Gin Lys Arg Leu Phe Gin Lys Tyr Gly Ile Asp Asn Pro Asp Met

245 250 255

Asn Lys Leu Gin Phe His Leu Met Leu Asp Glu Phe Phe 260 265

<210> 165

<211> 786 <212> DNA

<213> Bacillus

<220>

<221> CDS

<222> (1) . . (786)

<223> Essential gene sufC in the iscU operon

<400> 165 atg gct gct tca aca tta acg atc aaa gat ctt cac gtt gaa atc gaa 48

Met Ala Ala Ser Thr Leu Thr Ile Lys Asp Leu His Val Glu Ile Glu

1 5 10 15 ggg aaa gag atc tta aag ggt gta aac ctt gaa ata aaa ggt gga gaa 96

Gly Lys Glu Ile Leu Lys Gly Val Asn Leu Glu Ile Lys Gly Gly Glu

20 25 30 ttc cac gca gta atg ggc ccg aac gga act ggt aaa tec act tta tca 144

Phe His Ala Val Met Gly Pro Asn Gly Thr Gly Lys Ser Thr Leu Ser

35 40 45 gct gct att atg ggg cat cct aaa tat gaa gta aca aaa ggc agc atc 192

Ala Ala Ile Met Gly His Pro Lys Tyr Glu Val Thr Lys Gly Ser Ile

50 55 60 acg ctt gac ggc aaa gat gta ctg gag atg gaa gtg gac gag cgt gcg 240

Thr Leu Asp Gly Lys Asp Val Leu Glu Met Glu Val Asp Glu Arg Ala

65 70 75 80 cag gca ggc tta ttc ctt gca atg cag tac cca agt gaa atc agc ggt 288

Gin Ala Gly Leu Phe Leu Ala Met Gin Tyr Pro Ser Glu Ile Ser Gly

85 90 95 gtg aca aat gcc gac ttc ctt cgc tca gca atc aac gcg cgc aga gaa 336 Val Thr Asn Ala Asp Phe Leu Arg Ser Ala Ile Asn Ala Arg Arg Glu

100 105 110 gaa ggc gat gaa att tct ctc atg aaa ttc atc cgc aaa atg gac gaa 384

Glu Gly Asp Glu Ile Ser Leu Met Lys Phe Ile Arg Lys Met Asp Glu

115 120 125 aac atg gag ttc ctt gaa atg gac cct gaa atg gct cag cgc tat ctt 432

Asn Met Glu Phe Leu Glu Met Asp Pro Glu Met Ala Gin Arg Tyr Leu

130 135 140 aac gaa ggc ttc tca ggc ggg gag aaa aaa cgc aac gaa atc ctt caa 480

Asn Glu Gly Phe Ser Gly Gly Glu Lys Lys Arg Asn Glu Ile Leu Gin

145 150 155 160 tta atg atg att gaa ccg aaa atc gcc atc ctt gat gaa att gac tca 528

Leu Met Met Ile Glu Pro Lys Ile Ala Ile Leu Asp Glu Ile Asp Ser

165 170 175 ggc ctt gat att gac gct ttg aaa gtt gta tca aaa ggg atc aac aaa 576

Gly Leu Asp Ile Asp Ala Leu Lys Val Val Ser Lys Gly Ile Asn Lys

180 185 190 atg cgc agc gaa aac ttc ggc tgc ctg atg atc act cac tat cag cgc 624

Met Arg Ser Glu Asn Phe Gly Cys Leu Met Ile Thr His Tyr Gin Arg

195 200 205 ctg tta aac tac atc act ccg gat gtt gtt cac gtt atg atg caa ggc 672 Leu Leu Asn Tyr Ile Thr Pro Asp Val Val His Val Met Met Gin Gly 210 215 220 cgc gtt gta aaa tcc ggc ggt gca gag ctt gca cag cgt ctt gaa gca 720 Arg Val Val Lys Ser Gly Gly Ala Glu Leu Ala Gin Arg Leu Glu Ala 225 230 235 240 gaa ggt tat gac tgg atc aaa caa gaa ctt ggc att gaa gac gaa act 768 Glu Gly Tyr Asp Trp Ile Lys Gin Glu Leu Gly Ile Glu Asp Glu Thr 245 250 255 gtt ggc caa gaa gcg taa 786 Val Gly Gin Glu Ala 260

<210> 166

<211> 261

<212> PRT <213> Bacillus

<400> 166

Met Ala Ala Ser Thr Leu Thr Ile Lys Asp Leu His Val Glu Ile Glu 1 5 10 15

Gly Lys Glu Ile Leu Lys Gly Val Asn Leu Glu Ile Lys Gly Gly Glu 20 25 30

Phe His Ala Val Met Gly Pro Asn Gly Thr Gly Lys Ser Thr Leu Ser 35 40 45

Ala Ala Ile Met Gly His Pro Lys Tyr Glu Val Thr Lys Gly Ser Ile 50 55 60

Thr Leu Asp Gly Lys Asp Val Leu Glu Met Glu Val Asp Glu Arg Ala 65 70 75 80

Gin Ala Gly Leu Phe Leu Ala Met Gin Tyr Pro Ser Glu Ile Ser Gly 85 90 95 Val Thr Asn Ala Asp Phe Leu Arg Ser Ala Ile Asn Ala Arg Arg Glu

100 105 110

Glu Gly Asp Glu Ile Ser Leu Met Lys Phe Ile Arg Lys Met Asp Glu 115 120 125

Asn Met Glu Phe Leu Glu Met Asp Pro Glu Met Ala Gin Arg Tyr Leu 130 135 140

Asn Glu Gly Phe Ser Gly Gly Glu Lys Lys Arg Asn Glu Ile Leu Gin 145 150 155 160 Leu Met Met Ile Glu Pro Lys Ile Ala Ile Leu Asp Glu Ile Asp Ser 165 170 175

Gly Leu Asp Ile Asp Ala Leu Lys Val Val Ser Lys Gly Ile Asn Lys 180 185 190

Met Arg Ser Glu Asn Phe Gly Cys Leu Met Ile Thr His Tyr Gin Arg 195 200 205

Leu Leu Asn Tyr Ile Thr Pro Asp Val Val His Val Met Met Gin Gly

210 215 220

Arg Val Val Lys Ser Gly Gly Ala Glu Leu Ala Gin Arg Leu Glu Ala 225 230 235 240

Glu Gly Tyr Asp Trp Ile Lys Gin Glu Leu Gly Ile Glu Asp Glu Thr

245 250 255 Val Gly Gin Glu Ala 260

<210> 167

<211> 1314

<212> DNA

<213> Bacillus

<220>

<221> CDS

<222> (1) . . (1314 )

<223> Essential gene sufD in the iscU operon

<400> 167 atg aca cta ggt aca aaa cta tcc gta gat cag gag tat ctc aaa agc 48

Met Thr Leu Gly Thr Lys Leu Ser Val Asp Gin Glu Tyr Leu Lys Ser

1 5 10 15 ttt tcc gaa aag cac caa gaa cct gcc tgg ctg aag aac ctg cgc tta 96

Phe Ser Glu Lys His Gin Glu Pro Ala Trp Leu Lys Asn Leu Arg Leu

20 25 30 cag gct ctt gaa caa gct gag gat ctg ccg atg ccg aaa cct gac aaa 144

Gin Ala Leu Glu Gin Ala Glu Asp Leu Pro Met Pro Lys Pro Asp Lys

35 40 45 aca aaa atc aca aac tgg aac ttc acg aat ttc gcg aag cat aca gtg 192

Thr Lys Ile Thr Asn Trp Asn Phe Thr Asn Phe Ala Lys His Thr Val

50 55 60 gat aac gaa ccg ctt tct tca tta gaa gat ttg act gat gaa gta aaa 240

Asp Asn Glu Pro Leu Ser Ser Leu Glu Asp Leu Thr Asp Glu Val Lys

65 70 75 80 gcg ctg atc gac att gaa aac gaa gat aaa aca tta tac gta cag cgt 288

Ala Leu Ile Asp Ile Glu Asn Glu Asp Lys Thr Leu Tyr Val Gin Arg

85 90 95 gac cag acg ccg gca cat ctt tct ctt tct caa gaa ttg aaa gac aaa 336

Asp Gin Thr Pro Ala His Leu Ser Leu Ser Gin Glu Leu Lys Asp Lys

100 105 110 ggc gtc atc ttc act gat att ctg act gct gca cgc gag cac agc gac 384

Gly Val Ile Phe Thr Asp Ile Leu Thr Ala Ala Arg Glu His Ser Asp

115 120 125 ttg gtg gag aaa tac ttt atg aag gac ggc gtg aag gtt gac gaa cat 432

Leu Val Glu Lys Tyr Phe Met Lys Asp Gly Val Lys Val Asp Glu His

130 135 140 aag tta act gca ctt cac gcg gct ctt gtt aac gga gga gca ttc ctt 480

Lys Leu Thr Ala Leu His Ala Ala Leu Val Asn Gly Gly Ala Phe Leu

145 150 155 160 tat gtt ccg aaa aat gtt cag gtg gag acg ccg gtt cag gct gtt tat 528

Tyr Val Pro Lys Asn Val Gin Val Glu Thr Pro Val Gin Ala Val Tyr

165 170 175 gtc cac gaa agc aat gat aca gct cta ttc aac cac gtg ctg att gtg 576 Val His Glu Ser Asn Asp Thr Ala Leu Phe Asn His Val Leu Ile Val

180 185 190 gct gaa gac cac agc tct gta aca tat gta gaa aac tac atc agc act 624

Ala Glu Asp His Ser Ser Val Thr Tyr Val Glu Asn Tyr Ile Ser Thr

195 200 205 gta aac ccg aaa gac gct gtc ttc aat att atc agt gag gtc atc aca 672 Val Asn Pro Lys Asp Ala Val Phe Asn Ile Ile Ser Glu Val Ile Thr

210 215 220 ggt gat aat gcc agc gtg aca tac ggt gcg gtg gat aat ctg tca agc 720

Gly Asp Asn Ala Ser Val Thr Tyr Gly Ala Val Asp Asn Leu Ser Ser

225 230 235 240 ggt gtg aca act tat gtg aac cgc cgc ggt gct gcg cgc gga cgc gac 768

Gly Val Thr Thr Tyr Val Asn Arg Arg Gly Ala Ala Arg Gly Arg Asp

245 250 255 agc aaa atc gag tgg gct ctc ggc ctg atg aac gat ggc gac acg att 816

Ser Lys Ile Glu Trp Ala Leu Gly Leu Met Asn Asp Gly Asp Thr Ile

260 265 270 tcc gaa aac aca aca aac ctt tac ggt gac ggc aca tac ggc gat acg 864

Ser Glu Asn Thr Thr Asn Leu Tyr Gly Asp Gly Thr Tyr Gly Asp Thr

275 280 285 aaa acg gtt gtt gtc gga aga gga gag caa aca gaa aac ttc aca aca 912

Lys Thr Val Val Val Gly Arg Gly Glu Gin Thr Glu Asn Phe Thr Thr

290 295 300 caa atc atc cat ttc ggt aaa gct tct gaa ggc tat atc ttg aag cac 960

Gin Ile Ile His Phe Gly Lys Ala Ser Glu Gly Tyr Ile Leu Lys His

305 310 315 320 ggt gtg atg aag gat tcc gct tct tct atc ttt aac gga atc ggc aaa 1008

Gly Val Met Lys Asp Ser Ala Ser Ser Ile Phe Asn Gly Ile Gly Lys

325 330 335 atc gaa cat ggc gct tct aaa gcg aat gcg gag caa gaa tca cgc gtg 1056 Ile Glu His Gly Ala Ser Lys Ala Asn Ala Glu Gin Glu Ser Arg Val 340 345 350 ctg atg ctt agc gaa aaa gca cgc gga gac gca aac cct att ctt tta 1104

Leu Met Leu Ser Glu Lys Ala Arg Gly Asp Ala Asn Pro Ile Leu Leu

355 360 365 att gac gaa gac gat gta act gca gga cat gcg gca tct gtc ggc cgt 1152 Ile Asp Glu Asp Asp Val Thr Ala Gly His Ala Ala Ser Val Gly Arg

370 375 380 gtg gac cct att cag ctt tac tac ctg atg agc cgc gga att ccg aaa 1200 Val Asp Pro Ile Gin Leu Tyr Tyr Leu Met Ser Arg Gly Ile Pro Lys

385 390 395 400 gaa gaa gca gaa cgc tta gtc att tac gga ttc ctt gca ccg gta gta 1248

Glu Glu Ala Glu Arg Leu Val Ile Tyr Gly Phe Leu Ala Pro Val Val

405 410 415 aat gaa ctt ccg att gaa ggc gtt aag aaa caa tta gtt tct gtt atc 1296

Asn Glu Leu Pro Ile Glu Gly Val Lys Lys Gin Leu Val Ser Val Ile

420 425 430 gaa agg aaa gtg aag taa 1314

Glu Arg Lys Val Lys

435

<210> 168

<211> 437

<212> PRT

<213> Bacillus

<400> 168

Met Thr Leu Gly Thr Lys Leu Ser Val Asp Gin Glu Tyr Leu Lys Ser

1 5 10 15

Phe Ser Glu Lys His Gin Glu Pro Ala Trp Leu Lys Asn Leu Arg Leu 20 25 30

Gin Ala Leu Glu Gin Ala Glu Asp Leu Pro Met Pro Lys Pro Asp Lys 35 40 45

Thr Lys Ile Thr Asn Trp Asn Phe Thr Asn Phe Ala Lys His Thr Val 50 55 60

Asp Asn Glu Pro Leu Ser Ser Leu Glu Asp Leu Thr Asp Glu Val Lys 65 70 75 80

Ala Leu Ile Asp Ile Glu Asn Glu Asp Lys Thr Leu Tyr Val Gin Arg 85 90 95

Asp Gin Thr Pro Ala His Leu Ser Leu Ser Gin Glu Leu Lys Asp Lys 100 105 110

Gly Val Ile Phe Thr Asp Ile Leu Thr Ala Ala Arg Glu His Ser Asp 115 120 125

Leu Val Glu Lys Tyr Phe Met Lys Asp Gly Val Lys Val Asp Glu His 130 135 140

Lys Leu Thr Ala Leu His Ala Ala Leu Val Asn Gly Gly Ala Phe Leu

145 150 155 160

Tyr Val Pro Lys Asn Val Gin Val Glu Thr Pro Val Gin Ala Val Tyr 165 170 175 Val His Glu Ser Asn Asp Thr Ala Leu Phe Asn His Val Leu Ile Val 180 185 190

Ala Glu Asp His Ser Ser Val Thr Tyr Val Glu Asn Tyr Ile Ser Thr 195 200 205 Val Asn Pro Lys Asp Ala Val Phe Asn Ile Ile Ser Glu Val Ile Thr 210 215 220

Gly Asp Asn Ala Ser Val Thr Tyr Gly Ala Val Asp Asn Leu Ser Ser

225 230 235 240

Gly Val Thr Thr Tyr Val Asn Arg Arg Gly Ala Ala Arg Gly Arg Asp

245 250 255

Ser Lys Ile Glu Trp Ala Leu Gly Leu Met Asn Asp Gly Asp Thr Ile 260 265 270

Ser Glu Asn Thr Thr Asn Leu Tyr Gly Asp Gly Thr Tyr Gly Asp Thr 275 280 285

Lys Thr Val Val Val Gly Arg Gly Glu Gin Thr Glu Asn Phe Thr Thr 290 295 300

Gin Ile Ile His Phe Gly Lys Ala Ser Glu Gly Tyr Ile Leu Lys His

305 310 315 320

Gly Val Met Lys Asp Ser Ala Ser Ser Ile Phe Asn Gly Ile Gly Lys

325 330 335 Ile Glu His Gly Ala Ser Lys Ala Asn Ala Glu Gin Glu Ser Arg Val

340 345 350

Leu Met Leu Ser Glu Lys Ala Arg Gly Asp Ala Asn Pro Ile Leu Leu 355 360 365 Ile Asp Glu Asp Asp Val Thr Ala Gly His Ala Ala Ser Val Gly Arg

370 375 380 Val Asp Pro Ile Gin Leu Tyr Tyr Leu Met Ser Arg Gly Ile Pro Lys 385 390 395 400

Glu Glu Ala Glu Arg Leu Val Ile Tyr Gly Phe Leu Ala Pro Val Val

405 410 415

Asn Glu Leu Pro Ile Glu Gly Val Lys Lys Gin Leu Val Ser Val Ile 420 425 430

Glu Arg Lys Val Lys 435

<210> 169

<211> 1221

<212> DNA

<213> Bacilllus

<220>

<221> CDS

<222> (1) . . (1221)

<223> Essential gene sufS in the iscU operon

<400> 169 atg aat atc aca gat att cgt gaa cag ttc ccg atc ctt cat cag caa 48

Met Asn Ile Thr Asp Ile Arg Glu Gin Phe Pro Ile Leu His Gin Gin

1 5 10 15 gtg aac gga cat gat ctc gtt tat ctc gac agc gcg gcg act tcc cag 96 Val Asn Gly His Asp Leu Val Tyr Leu Asp Ser Ala Ala Thr Ser Gin

20 25 30 aag ccg cgt gct gtc att gaa aca ctg gat aag tat tat aac caa tac 144

Lys Pro Arg Ala Val Ile Glu Thr Leu Asp Lys Tyr Tyr Asn Gin Tyr

35 40 45 aat tcc aat gtt cac egg ggc gtc cat aca ctt gga acc aga gcg aca 192

Asn Ser Asn Val His Arg Gly Val His Thr Leu Gly Thr Arg Ala Thr

50 55 60 gat ggt tat gaa gga gcg cgt gaa aaa gtc cgc aag ttt att aac gcg 240

Asp Gly Tyr Glu Gly Ala Arg Glu Lys Val Arg Lys Phe Ile Asn Ala

65 70 75 80 aag tca atg gct gag att att ttc aca aaa ggc acg acc aca tca ctg 288

Lys Ser Met Ala Glu Ile Ile Phe Thr Lys Gly Thr Thr Thr Ser Leu

85 90 95 aat atg gtg gcg tta agc tat gcg cgc gcc aac ctg aaa cct ggt gat 336

Asn Met Val Ala Leu Ser Tyr Ala Arg Ala Asn Leu Lys Pro Gly Asp

100 105 110 gaa gtg gtc atc acc tac atg gag cat cat gcg aat atc att ccg tgg 384

Glu Val Val Ile Thr Tyr Met Glu His His Ala Asn Ile Ile Pro Trp

115 120 125 cag cag gct gta aaa gca act ggc gcc aca tta aaa tat att ccg ttg 432

Gin Gin Ala Val Lys Ala Thr Gly Ala Thr Leu Lys Tyr Ile Pro Leu

130 135 140 cag gaa gac gga acg att tct tta gaa gac gtc aga gaa acg gtc aca 480

Gin Glu Asp Gly Thr Ile Ser Leu Glu Asp Val Arg Glu Thr Val Thr

145 150 155 160 agc aac aca aaa att gtt gcg gtg tct cat gta tct aat gtt ctc ggc 528

Ser Asn Thr Lys Ile Val Ala Val Ser His Val Ser Asn Val Leu Gly

165 170 175 acg gtc aac ccg att aaa gaa atg gcg aaa atc gct cat gac aat ggg 576

Thr Val Asn Pro Ile Lys Glu Met Ala Lys Ile Ala His Asp Asn Gly

180 185 190 gct gtc att gtg gtg gac ggt gca caa agc acg cct cac atg aaa att 624

Ala Val Ile Val Val Asp Gly Ala Gin Ser Thr Pro His Met Lys Ile

195 200 205 gac gtg cag gat ctc gat tgc gac ttc ttt gcg ctt tct tcc cat aaa 672

Asp Val Gin Asp Leu Asp Cys Asp Phe Phe Ala Leu Ser Ser His Lys

210 215 220 atg tgc gga cct acc ggt gtt ggt gtg ctg tac gga aag aaa gcc ctg 720

Met Cys Gly Pro Thr Gly Val Gly Val Leu Tyr Gly Lys Lys Ala Leu

225 230 235 240 ctg gaa aac atg gag cct gcc gaa ttc ggc ggt gaa atg atc gac ttt 768

Leu Glu Asn Met Glu Pro Ala Glu Phe Gly Gly Glu Met Ile Asp Phe

245 250 255 gtc ggg ctt tat gaa tca act tgg aaa gag ctt ccg tgg aaa ttc gaa 816 Val Gly Leu Tyr Glu Ser Thr Trp Lys Glu Leu Pro Trp Lys Phe Glu

260 265 270 gcg ggt acg ccg att att gca gga gca atc ggc ctc ggt gcc gcc att 864

Ala Gly Thr Pro Ile Ile Ala Gly Ala Ile Gly Leu Gly Ala Ala Ile

275 280 285 gat ttt ctc gaa gaa atc ggt ctt gat gag att tcc cgc cat gag cac 912

Asp Phe Leu Glu Glu Ile Gly Leu Asp Glu Ile Ser Arg His Glu His

290 295 300 aag ctt gca gct tat gcg ctt gaa cgc ttc cgc cag ctt gac ggc gta 960

Lys Leu Ala Ala Tyr Ala Leu Glu Arg Phe Arg Gin Leu Asp Gly Val

305 310 315 320 acg gtt tac ggg ccg gaa gag cgt gcc ggg ctt gtc aca ttt aac ctt 1008

Thr Val Tyr Gly Pro Glu Glu Arg Ala Gly Leu Val Thr Phe Asn Leu

325 330 335 gat gat gtt cat ccg cac gat gtg gcg act gtg ctt gat gcg gag gga 1056

Asp Asp Val His Pro His Asp Val Ala Thr Val Leu Asp Ala Glu Gly

340 345 350 atc gca gtc aga gcc ggc cac cat tgt gcg cag ccg ctg atg aaa tgg 1104 Ile Ala Val Arg Ala Gly His His Cys Ala Gin Pro Leu Met Lys Trp

355 360 365 ctg gat gta act gct act gca aga gca agc ttt tat ctg tat aat aca 1152

Leu Asp Val Thr Ala Thr Ala Arg Ala Ser Phe Tyr Leu Tyr Asn Thr 370 375 380 gag gaa gag att gat aag ctt gtg gaa gct ctt caa aag aca aag gag 1200 Glu Glu Glu Ile Asp Lys Leu Val Glu Ala Leu Gin Lys Thr Lys Glu 385 390 395 400 tat ttt aca aat gtc ttt taa 1221 Tyr Phe Thr Asn Val Phe

405

<210> 170

<211> 406

<212> PRT

<213> Bacilllus

<400> 170

Met Asn Ile Thr Asp Ile Arg Glu Gin Phe Pro Ile Leu His Gin Gin

1 5 10 15 Val Asn Gly His Asp Leu Val Tyr Leu Asp Ser Ala Ala Thr Ser Gin 20 25 30

Lys Pro Arg Ala Val Ile Glu Thr Leu Asp Lys Tyr Tyr Asn Gin Tyr 35 40 45

Asn Ser Asn Val His Arg Gly Val His Thr Leu Gly Thr Arg Ala Thr

50 55 60

Asp Gly Tyr Glu Gly Ala Arg Glu Lys Val Arg Lys Phe Ile Asn Ala 65 70 75 80

Lys Ser Met Ala Glu Ile Ile Phe Thr Lys Gly Thr Thr Thr Ser Leu 85 90 95

Asn Met Val Ala Leu Ser Tyr Ala Arg Ala Asn Leu Lys Pro Gly Asp 100 105 110

Glu Val Val Ile Thr Tyr Met Glu His His Ala Asn Ile Ile Pro Trp

115 120 125

Gin Gin Ala Val Lys Ala Thr Gly Ala Thr Leu Lys Tyr Ile Pro Leu

130 135 140

Gin Glu Asp Gly Thr Ile Ser Leu Glu Asp Val Arg Glu Thr Val Thr 145 150 155 160

Ser Asn Thr Lys Ile Val Ala Val Ser His Val Ser Asn Val Leu Gly 165 170 175

Thr Val Asn Pro Ile Lys Glu Met Ala Lys Ile Ala His Asp Asn Gly 180 185 190

Ala Val Ile Val Val Asp Gly Ala Gin Ser Thr Pro His Met Lys Ile 195 200 205

Asp Val Gin Asp Leu Asp Cys Asp Phe Phe Ala Leu Ser Ser His Lys

210 215 220

Met Cys Gly Pro Thr Gly Val Gly Val Leu Tyr Gly Lys Lys Ala Leu 225 230 235 240

Leu Glu Asn Met Glu Pro Ala Glu Phe Gly Gly Glu Met Ile Asp Phe

245 250 255 Val Gly Leu Tyr Glu Ser Thr Trp Lys Glu Leu Pro Trp Lys Phe Glu

260 265 270

Ala Gly Thr Pro Ile Ile Ala Gly Ala Ile Gly Leu Gly Ala Ala Ile 275 280 285

Asp Phe Leu Glu Glu Ile Gly Leu Asp Glu Ile Ser Arg His Glu His

290 295 300

Lys Leu Ala Ala Tyr Ala Leu Glu Arg Phe Arg Gin Leu Asp Gly Val 305 310 315 320

Thr Val Tyr Gly Pro Glu Glu Arg Ala Gly Leu Val Thr Phe Asn Leu 325 330 335

Asp Asp Val His Pro His Asp Val Ala Thr Val Leu Asp Ala Glu Gly

340 345 350 Ile Ala Val Arg Ala Gly His His Cys Ala Gin Pro Leu Met Lys Trp

355 360 365

Leu Asp Val Thr Ala Thr Ala Arg Ala Ser Phe Tyr Leu Tyr Asn Thr

370 375 380

Glu Glu Glu Ile Asp Lys Leu Val Glu Ala Leu Gin Lys Thr Lys Glu 385 390 395 400

Tyr Phe Thr Asn Val Phe

405

<210> 171

<211> 444

<212> DNA

<213> Bacillus <220>

<221> CDS

<222> (1) . . (444 )

<223> Essential gene sufU (synonym iscU) in the iscU operon

<400> 171 atg tct ttt aat gca aac tta gat aca ttg tac aga cag gtg att atg 48

Met Ser Phe Asn Ala Asn Leu Asp Thr Leu Tyr Arg Gin Val Ile Met 1 5 10 15 gat cat tac aaa aac ccg cga aat aaa ggg gtt tta aat gac agc att 96

Asp His Tyr Lys Asn Pro Arg Asn Lys Gly Val Leu Asn Asp Ser Ile

20 25 30 gtc gtg gat atg aac aat ccg aca tgc ggc gac cgc atc aga ctg aca 144 Val Val Asp Met Asn Asn Pro Thr Cys Gly Asp Arg Ile Arg Leu Thr

35 40 45 atg aag ctt gac gga gac atc gtg gaa gat gcg aag ttt gaa ggg gaa 192

Met Lys Leu Asp Gly Asp Ile Val Glu Asp Ala Lys Phe Glu Gly Glu

50 55 60 ggc tgt tcc att tca atg gca tec gct tcg atg atg act cag gcg att 240

Gly Cys Ser Ile Ser Met Ala Ser Ala Ser Met Met Thr Gin Ala Ile

65 70 75 80 aaa ggg aaa gat att gaa act gcc ctt tcc atg tct aag att ttc tcg 288

Lys Gly Lys Asp Ile Glu Thr Ala Leu Ser Met Ser Lys Ile Phe Ser

85 90 95 gat atg atg caa ggg aaa gag tat gat gat tct ata gat ctc ggg gat 336

Asp Met Met Gin Gly Lys Glu Tyr Asp Asp Ser Ile Asp Leu Gly Asp

100 105 110 att gaa gcc ctt caa ggc gtt tca aaa ttc cct gcc cgt atc aaa tgt 384 Ile Glu Ala Leu Gin Gly Val Ser Lys Phe Pro Ala Arg Ile Lys Cys

115 120 125 gca acc ctg tca tgg aaa gca ctt gaa aaa gga gtc gcg aaa gaa gaa 432

Ala Thr Leu Ser Trp Lys Ala Leu Glu Lys Gly Val Ala Lys Glu Glu

130 135 140 ggc ggc aat taa 444

Gly Gly Asn 145

<210> 172

<211> 147

<212> PRT

<213> Bacillus

<400> 172

Met Ser Phe Asn Ala Asn Leu Asp Thr Leu Tyr Arg Gin Val Ile Met

1 5 10 15

Asp His Tyr Lys Asn Pro Arg Asn Lys Gly Val Leu Asn Asp Ser Ile 20 25 30 Val Val Asp Met Asn Asn Pro Thr Cys Gly Asp Arg Ile Arg Leu Thr 35 40 45

Met Lys Leu Asp Gly Asp Ile Val Glu Asp Ala Lys Phe Glu Gly Glu

50 55 60

Gly Cys Ser Ile Ser Met Ala Ser Ala Ser Met Met Thr Gin Ala Ile 65 70 75 80

Lys Gly Lys Asp Ile Glu Thr Ala Leu Ser Met Ser Lys Ile Phe Ser 85 90 95

Asp Met Met Gin Gly Lys Glu Tyr Asp Asp Ser Ile Asp Leu Gly Asp 100 105 110 Ile Glu Ala Leu Gin Gly Val Ser Lys Phe Pro Ala Arg Ile Lys Cys

115 120 125

Ala Thr Leu Ser Trp Lys Ala Leu Glu Lys Gly Val Ala Lys Glu Glu

130 135 140

Gly Gly Asn 145

<210> 173

<211> 1398

<212> DNA

<213> Bacillus

<220>

<221> CDS

<222> (1) . . (1398)

<223> Essential gene sufB in the iscU operon

<400> 173 atg gct aaa aaa atg cct gat att ggt gaa tac aag tat ggt ttt cac 48

Met Ala Lys Lys Met Pro Asp Ile Gly Glu Tyr Lys Tyr Gly Phe His 1 5 10 15 gac aag gac gtt tcc att ttc cgt tca gag cgc gga ttg aca aaa gaa 96

Asp Lys Asp Val Ser Ile Phe Arg Ser Glu Arg Gly Leu Thr Lys Glu

20 25 30 atc gta gaa gaa att tct cgc atg aaa gaa gag cct caa tgg atg ctt 144 Ile Val Glu Glu Ile Ser Arg Met Lys Glu Glu Pro Gin Trp Met Leu

35 40 45 gac ttc cgc ttg aaa tca ctt gag cat ttc tac aat atg ccg atg cct 192

Asp Phe Arg Leu Lys Ser Leu Glu His Phe Tyr Asn Met Pro Met Pro

50 55 60 caa tgg ggc gga gat tta aac tca ctg aac ttt gac gaa att acg tac 240

Gin Trp Gly Gly Asp Leu Asn Ser Leu Asn Phe Asp Glu Ile Thr Tyr 65 70 75 80 tac gta aaa ccg tct gag cgt tca gag cgt tct tgg gat gaa gtt cct 288

Tyr Val Lys Pro Ser Glu Arg Ser Glu Arg Ser Trp Asp Glu Val Pro

85 90 95 gaa gaa att aaa caa aca ttc gac aag ctc ggt att cct gaa gct gaa 336

Glu Glu Ile Lys Gin Thr Phe Asp Lys Leu Gly Ile Pro Glu Ala Glu

100 105 110 caa aaa tac ctt gcg ggt gtt tct gct cag tac gaa tct gag gtt gta 384

Gin Lys Tyr Leu Ala Gly Val Ser Ala Gin Tyr Glu Ser Glu Val Val

115 120 125 tac cac aac atg aaa gaa gat ctt gaa gca caa ggc atc gtc ttt aaa 432

Tyr His Asn Met Lys Glu Asp Leu Glu Ala Gin Gly Ile Val Phe Lys

130 135 140 gac act gac agc gcg ctg aaa gag aat gaa gat att ttc cgt gag cac 480

Asp Thr Asp Ser Ala Leu Lys Glu Asn Glu Asp Ile Phe Arg Glu His

145 150 155 160 tgg gca aaa gta atc ccg ccg act gat aac aag ttt gcg gcg ctt aac 528

Trp Ala Lys Val Ile Pro Pro Thr Asp Asn Lys Phe Ala Ala Leu Asn

165 170 175 tca gct gtt tgg tcc ggc ggt tct ttc atc tac gtg cct aaa ggc gtg 576

Ser Ala Val Trp Ser Gly Gly Ser Phe Ile Tyr Val Pro Lys Gly Val

180 185 190 aag gtt gaa aca ccg cta caa gct tac ttc cgt atc aac tct gag aac 624

Lys Val Glu Thr Pro Leu Gin Ala Tyr Phe Arg Ile Asn Ser Glu Asn

195 200 205 atg ggt cag ttc gaa cgt aca ctg atc att gtt gac gaa gaa gca agc 672

Met Gly Gin Phe Glu Arg Thr Leu Ile Ile Val Asp Glu Glu Ala Ser

210 215 220 gtt cat tat gtt gaa ggc tgt act gcg cct gtt tac aca aca aac tca 720 Val His Tyr Val Glu Gly Cys Thr Ala Pro Val Tyr Thr Thr Asn Ser

225 230 235 240 ctt cac agt gcg gtc gtt gag atc atc gtg aaa aaa ggc ggc tac tgc 768

Leu His Ser Ala Val Val Glu Ile Ile Val Lys Lys Gly Gly Tyr Cys

245 250 255 cgt tat aca acg atc caa aac tgg gcg aac aac gtt tac aac ctt gta 816

Arg Tyr Thr Thr Ile Gin Asn Trp Ala Asn Asn Val Tyr Asn Leu Val

260 265 270 aca aag cgt aca gtc tgt gaa gaa aac gca aca atg gaa tgg atc gac 864

Thr Lys Arg Thr Val Cys Glu Glu Asn Ala Thr Met Glu Trp Ile Asp

275 280 285 ggc aac atc ggt tct aag ctg aca atg aaa tat ccg gct tgt atc cta 912

Gly Asn Ile Gly Ser Lys Leu Thr Met Lys Tyr Pro Ala Cys Ile Leu

290 295 300 aaa ggc gaa ggc gcg cgc ggt atg acg ctt tct atc gct ctt gcg ggt 960

Lys Gly Glu Gly Ala Arg Gly Met Thr Leu Ser Ile Ala Leu Ala Gly

305 310 315 320 aaa ggc cag cat cag gat gca ggt gcg aaa atg att cac ctt gca cca 1008 Lys Gly Gin His Gin Asp Ala Gly Ala Lys Met Ile His Leu Ala Pro

325 330 335 aac aca tca tct aca atc gta tca aaa tcg att tca aaa cag ggc gga 1056

Asn Thr Ser Ser Thr Ile Val Ser Lys Ser Ile Ser Lys Gin Gly Gly

340 345 350 aaa gta acg tac cgc gga atc gtc cac ttc gga cgc aaa gcg gaa ggc 1104

Lys Val Thr Tyr Arg Gly Ile Val His Phe Gly Arg Lys Ala Glu Gly

355 360 365 gcc cgc tct aac atc gag tgt gat acg ctc att atg gat aac aaa tca 1152

Ala Arg Ser Asn Ile Glu Cys Asp Thr Leu Ile Met Asp Asn Lys Ser

370 375 380 act tct gac aca att cct tac aat gaa atc tta aat gat aac att tca 1200

Thr Ser Asp Thr Ile Pro Tyr Asn Glu Ile Leu Asn Asp Asn Ile Ser

385 390 395 400 ttg gag cac gaa gcg aaa gtg tct aaa gta tct gaa gag cag ctc ttc 1248

Leu Glu His Glu Ala Lys Val Ser Lys Val Ser Glu Glu Gin Leu Phe

405 410 415 tac ttg atg agc cgc ggt att tct gaa gaa gaa gca act gaa atg atc 1296

Tyr Leu Met Ser Arg Gly Ile Ser Glu Glu Glu Ala Thr Glu Met Ile

420 425 430 gta atg ggc ttt atc gag cca ttc aca aaa gag ctt ccg atg gaa tac 1344 Val Met Gly Phe Ile Glu Pro Phe Thr Lys Glu Leu Pro Met Glu Tyr

435 440 445 gca gtt gaa atg aac cgt ttg att aag ttc gaa atg gaa ggt tct atc 1392

Ala Val Glu Met Asn Arg Leu Ile Lys Phe Glu Met Glu Gly Ser Ile

450 455 460 ggt taa 1398

Gly 465

<210> 174

<211> 465

<212> PRT

<213> Bacillus

<400> 174

Met Ala Lys Lys Met Pro Asp Ile Gly Glu Tyr Lys Tyr Gly Phe His 1 5 10 15

Asp Lys Asp Val Ser Ile Phe Arg Ser Glu Arg Gly Leu Thr Lys Glu 20 25 30 Ile Val Glu Glu Ile Ser Arg Met Lys Glu Glu Pro Gin Trp Met Leu 35 40 45

Asp Phe Arg Leu Lys Ser Leu Glu His Phe Tyr Asn Met Pro Met Pro 50 55 60 Gin Trp Gly Gly Asp Leu Asn Ser Leu Asn Phe Asp Glu Ile Thr Tyr 65 70 75 80

Tyr Val Lys Pro Ser Glu Arg Ser Glu Arg Ser Trp Asp Glu Val Pro 85 90 95

Glu Glu Ile Lys Gin Thr Phe Asp Lys Leu Gly Ile Pro Glu Ala Glu 100 105 110

Gin Lys Tyr Leu Ala Gly Val Ser Ala Gin Tyr Glu Ser Glu Val Val 115 120 125

Tyr His Asn Met Lys Glu Asp Leu Glu Ala Gin Gly Ile Val Phe Lys

130 135 140

Asp Thr Asp Ser Ala Leu Lys Glu Asn Glu Asp Ile Phe Arg Glu His 145 150 155 160

Trp Ala Lys Val Ile Pro Pro Thr Asp Asn Lys Phe Ala Ala Leu Asn

165 170 175

Ser Ala Val Trp Ser Gly Gly Ser Phe Ile Tyr Val Pro Lys Gly Val 180 185 190

Lys Val Glu Thr Pro Leu Gin Ala Tyr Phe Arg Ile Asn Ser Glu Asn 195 200 205

Met Gly Gin Phe Glu Arg Thr Leu Ile Ile Val Asp Glu Glu Ala Ser

210 215 220 Val His Tyr Val Glu Gly Cys Thr Ala Pro Val Tyr Thr Thr Asn Ser 225 230 235 240

Leu His Ser Ala Val Val Glu Ile Ile Val Lys Lys Gly Gly Tyr Cys

245 250 255

Arg Tyr Thr Thr Ile Gin Asn Trp Ala Asn Asn Val Tyr Asn Leu Val 260 265 270

Thr Lys Arg Thr Val Cys Glu Glu Asn Ala Thr Met Glu Trp Ile Asp 275 280 285

Gly Asn Ile Gly Ser Lys Leu Thr Met Lys Tyr Pro Ala Cys Ile Leu

290 295 300

Lys Gly Glu Gly Ala Arg Gly Met Thr Leu Ser Ile Ala Leu Ala Gly 305 310 315 320 Lys Gly Gin His Gin Asp Ala Gly Ala Lys Met Ile His Leu Ala Pro

325 330 335

Asn Thr Ser Ser Thr Ile Val Ser Lys Ser Ile Ser Lys Gin Gly Gly 340 345 350

Lys Val Thr Tyr Arg Gly Ile Val His Phe Gly Arg Lys Ala Glu Gly 355 360 365

Ala Arg Ser Asn Ile Glu Cys Asp Thr Leu Ile Met Asp Asn Lys Ser

370 375 380

Thr Ser Asp Thr Ile Pro Tyr Asn Glu Ile Leu Asn Asp Asn Ile Ser 385 390 395 400

Leu Glu His Glu Ala Lys Val Ser Lys Val Ser Glu Glu Gin Leu Phe

405 410 415

Tyr Leu Met Ser Arg Gly Ile Ser Glu Glu Glu Ala Thr Glu Met Ile 420 425 430 Val Met Gly Phe Ile Glu Pro Phe Thr Lys Glu Leu Pro Met Glu Tyr

435 440 445

Ala Val Glu Met Asn Arg Leu Ile Lys Phe Glu Met Glu Gly Ser Ile

450 455 460

Gly 465

<210> 175

<211> 480

<212> DNA

<213> Bacillus

<220>

<221> CDS

<222> (1) . . (480)

<223> Essential gene accB (in accC operon)

<400> 175 atg tta aat atc aaa gaa atc cac gag ctg att aaa gca att gac gag 48

Met Leu Asn Ile Lys Glu Ile His Glu Leu Ile Lys Ala Ile Asp Glu

1 5 10 15 tct aca att gac gaa ttc gta tat gaa aat gaa ggt gta tcc tta aaa 96

Ser Thr Ile Asp Glu Phe Val Tyr Glu Asn Glu Gly Val Ser Leu Lys

20 25 30 ctg aaa aaa cac gaa gca ggc acg gtt caa gtc atg cag cag gca ccg 144

Leu Lys Lys His Glu Ala Gly Thr Val Gin Val Met Gin Gin Ala Pro 35 40 45 gca gca cct gta caa gca cag gct ccg cag gca gtt cag ccg caa gcg 192 Ala Ala Pro Val Gin Ala Gin Ala Pro Gin Ala Val Gin Pro Gin Ala 50 55 60 cag caa gca gcg gca cct gcc caa gaa gca cca aag caa gat gag aat 240 Gin Gin Ala Ala Ala Pro Ala Gin Glu Ala Pro Lys Gin Asp Glu Asn 65 70 75 80 ctg cat aaa atc act tca cca atg gta gga aca ttt tat gct tct tca 288

Leu His Lys Ile Thr Ser Pro Met Val Gly Thr Phe Tyr Ala Ser Ser 85 90 95 tca ccg gaa gct ggc ccg tat gta aca gcc ggt tca aaa gta aat gaa 336

Ser Pro Glu Ala Gly Pro Tyr Val Thr Ala Gly Ser Lys Val Asn Glu

100 105 110 aac aca gtt gtc tgc att gta gaa gcg atg aag ctt ttc aac gaa atc 384

Asn Thr Val Val Cys Ile Val Glu Ala Met Lys Leu Phe Asn Glu Ile 115 120 125 gaa gca gaa gtg aaa ggc gaa atc gtt gaa gta tta gta gaa aac ggc 432

Glu Ala Glu Val Lys Gly Glu Ile Val Glu Val Leu Val Glu Asn Gly 130 135 140 cag ctg gtc gaa tac gga caa cct cta ttt ctt gta aaa gcg gag taa 480 Gin Leu Val Glu Tyr Gly Gin Pro Leu Phe Leu Val Lys Ala Glu 145 150 155

<210> 176

<211> 159

<212> PRT

<213> Bacillus

<400> 176

Met Leu Asn Ile Lys Glu Ile His Glu Leu Ile Lys Ala Ile Asp Glu

1 5 10 15

Ser Thr Ile Asp Glu Phe Val Tyr Glu Asn Glu Gly Val Ser Leu Lys 20 25 30

Leu Lys Lys His Glu Ala Gly Thr Val Gin Val Met Gin Gin Ala Pro 35 40 45

Ala Ala Pro Val Gin Ala Gin Ala Pro Gin Ala Val Gin Pro Gin Ala

50 55 60

Gin Gin Ala Ala Ala Pro Ala Gin Glu Ala Pro Lys Gin Asp Glu Asn 65 70 75 80

Leu His Lys Ile Thr Ser Pro Met Val Gly Thr Phe Tyr Ala Ser Ser 85 90 95

Ser Pro Glu Ala Gly Pro Tyr Val Thr Ala Gly Ser Lys Val Asn Glu 100 105 110

Asn Thr Val Val Cys Ile Val Glu Ala Met Lys Leu Phe Asn Glu Ile 115 120 125

Glu Ala Glu Val Lys Gly Glu Ile Val Glu Val Leu Val Glu Asn Gly

130 135 140

Gin Leu Val Glu Tyr Gly Gin Pro Leu Phe Leu Val Lys Ala Glu 145 150 155

<210> 177

<211> 1353

<212> DNA

<213> Bacillus

<220>

<221> CDS

<222> (1) . . (1353)

<223> Essential gene accC (in accC operon)

<400> 177 atg att aaa aag cta ttg atc gcc aac aga gga gaa att gct gtc aga 48

Met Ile Lys Lys Leu Leu Ile Ala Asn Arg Gly Glu Ile Ala Val Arg

1 5 10 15 atc atc aga gcc tgc aga gag ctc gga att gag act gtc gct gtt tat 96 Ile Ile Arg Ala Cys Arg Glu Leu Gly Ile Glu Thr Val Ala Val Tyr

20 25 30 tca gaa gct gat aaa gat gcc ctt cat gtt caa atg gcc gat gaa gct 144 Ser Glu Ala Asp Lys Asp Ala Leu His Val Gin Met Ala Asp Glu Ala 35 40 45 ttt tgt atc gga ccg aaa gca tca aaa gac agc tat tta aac gtt aca 192

Phe Cys Ile Gly Pro Lys Ala Ser Lys Asp Ser Tyr Leu Asn Val Thr

50 55 60 aat att gtg agt gtt gca aag ctg act ggc acg gac gcc att cat ccg 240

Asn Ile Val Ser Val Ala Lys Leu Thr Gly Thr Asp Ala Ile His Pro

65 70 75 80 gga tac gga ttt tta gct gaa aat gct gat ttc gct gaa tta tgt gaa 288 Gly Tyr Gly Phe Leu Ala Glu Asn Ala Asp Phe Ala Glu Leu Cys Glu 85 90 95 gaa gtg aat gtc acg ttt gtc ggc ccg agc gct gac gcc att tca aaa 336 Glu Val Asn Val Thr Phe Val Gly Pro Ser Ala Asp Ala Ile Ser Lys 100 105 110 atg gga aca aaa gac gtt gcg egg gaa acg atg aaa cag gcc ggc gtg 384

Met Gly Thr Lys Asp Val Ala Arg Glu Thr Met Lys Gin Ala Gly Val 115 120 125 cca atc gta ccg ggt tca cag gga att ata gaa aat gtg gaa gaa gcg 432 Pro Ile Val Pro Gly Ser Gin Gly Ile Ile Glu Asn Val Glu Glu Ala 130 135 140 gtt tcg ctt gct aat gaa att ggg tat cct gta att ata aaa gcc acc 480 Val Ser Leu Ala Asn Glu Ile Gly Tyr Pro Val Ile Ile Lys Ala Thr 145 150 155 160 gca ggc gga ggc gga aaa gga att agg gtt gcc cgc act gaa gag gaa 528

Ala Gly Gly Gly Gly Lys Gly Ile Arg Val Ala Arg Thr Glu Glu Glu

165 170 175 ctg att aat ggc att aag att aca cag cag gaa gcg gca act gca ttt 576

Leu Ile Asn Gly Ile Lys Ile Thr Gin Gin Glu Ala Ala Thr Ala Phe

180 185 190 ggg aat cca ggt gta tac atc gaa aaa tac ata gaa gat ttt cgc cac 624

Gly Asn Pro Gly Val Tyr Ile Glu Lys Tyr Ile Glu Asp Phe Arg His

195 200 205 gtt gag atc caa gtg ctt gct gat aac tac gga aat acg atc cat ttg 672 Val Glu Ile Gin Val Leu Ala Asp Asn Tyr Gly Asn Thr Ile His Leu

210 215 220 ggc gaa cgc gac tgc tcg atc caa aga cgc ctg caa aag ctt ttg gaa 720

Gly Glu Arg Asp Cys Ser Ile Gin Arg Arg Leu Gin Lys Leu Leu Glu

225 230 235 240 gaa tca cca tct cct gcc ctt gat tca gaa atc agg gag caa atg gga 768

Glu Ser Pro Ser Pro Ala Leu Asp Ser Glu Ile Arg Glu Gin Met Gly

245 250 255 gat gca gcg gta aag gct gcc aaa gcg gtt ggc tat aca ggt gct gga 816

Asp Ala Ala Val Lys Ala Ala Lys Ala Val Gly Tyr Thr Gly Ala Gly

260 265 270 aca gtt gaa ttt atc tat gac tac aat gaa cag cgc tat tac ttc atg 864

Thr Val Glu Phe Ile Tyr Asp Tyr Asn Glu Gin Arg Tyr Tyr Phe Met

275 280 285 gaa atg aac acg aga atc cag gtg gag cac cca gtc aca gaa atg gtg 912

Glu Met Asn Thr Arg Ile Gin Val Glu His Pro Val Thr Glu Met Val

290 295 300 acg gga act gac ctg atc aag gaa caa atc aaa gtg gct tca gga atg 960

Thr Gly Thr Asp Leu Ile Lys Glu Gin Ile Lys Val Ala Ser Gly Met

305 310 315 320 gaa ctg agc ctc aag caa gaa gat gtt gaa ttt gaa ggc tgg gcc atc 1008

Glu Leu Ser Leu Lys Gin Glu Asp Val Glu Phe Glu Gly Trp Ala Ile

325 330 335 gaa tgc cga atc aac gca gaa aac cca agc aaa aat ttc atg ccg tca 1056

Glu Cys Arg Ile Asn Ala Glu Asn Pro Ser Lys Asn Phe Met Pro Ser

340 345 350 cct ggt gaa att aaa atg tac ctg cct cct ggc ggt ctt ggt gtc cgt 1104

Pro Gly Glu Ile Lys Met Tyr Leu Pro Pro Gly Gly Leu Gly Val Arg

355 360 365 gtc gat tca gct gcg tac cct ggc tat tcc atc cca ccg tac tat gac 1152 Val Asp Ser Ala Ala Tyr Pro Gly Tyr Ser Ile Pro Pro Tyr Tyr Asp

370 375 380 agc atg att gca aag gta atc aca tac gga aaa acg aga gat gaa gcg 1200

Ser Met Ile Ala Lys Val Ile Thr Tyr Gly Lys Thr Arg Asp Glu Ala

385 390 395 400 att gcc cgc atg aag cgc gca ttg agc gaa ttc gtc atc gaa ggc att 1248 Ile Ala Arg Met Lys Arg Ala Leu Ser Glu Phe Val Ile Glu Gly Ile 405 410 415 gag aca aca atc cct ttc cat tta aaa ctg ctt gaa cac gaa aca ttt 1296 Glu Thr Thr Ile Pro Phe His Leu Lys Leu Leu Glu His Glu Thr Phe 420 425 430 gtg agc gga gag ttt aat acg aaa ttt tta gaa aca tat gat gta atg 1344 Val Ser Gly Glu Phe Asn Thr Lys Phe Leu Glu Thr Tyr Asp Val Met 435 440 445 ggc tca taa 1353

Gly Ser

450

<210> 178

<211> 450

<212> PRT

<213> Bacillus

<400> 178

Met Ile Lys Lys Leu Leu Ile Ala Asn Arg Gly Glu Ile Ala Val Arg

1 5 10 15 Ile Ile Arg Ala Cys Arg Glu Leu Gly Ile Glu Thr Val Ala Val Tyr 20 25 30

Ser Glu Ala Asp Lys Asp Ala Leu His Val Gin Met Ala Asp Glu Ala 35 40 45

Phe Cys Ile Gly Pro Lys Ala Ser Lys Asp Ser Tyr Leu Asn Val Thr 50 55 60

Asn Ile Val Ser Val Ala Lys Leu Thr Gly Thr Asp Ala Ile His Pro 65 70 75 80

Gly Tyr Gly Phe Leu Ala Glu Asn Ala Asp Phe Ala Glu Leu Cys Glu 85 90 95

Glu Val Asn Val Thr Phe Val Gly Pro Ser Ala Asp Ala Ile Ser Lys 100 105 110

Met Gly Thr Lys Asp Val Ala Arg Glu Thr Met Lys Gin Ala Gly Val 115 120 125

Pro Ile Val Pro Gly Ser Gin Gly Ile Ile Glu Asn Val Glu Glu Ala

130 135 140 Val Ser Leu Ala Asn Glu Ile Gly Tyr Pro Val Ile Ile Lys Ala Thr 145 150 155 160 Ala Gly Gly Gly Gly Lys Gly Ile Arg Val Ala Arg Thr Glu Glu Glu 165 170 175

Leu Ile Asn Gly Ile Lys Ile Thr Gin Gin Glu Ala Ala Thr Ala Phe

180 185 190

Gly Asn Pro Gly Val Tyr Ile Glu Lys Tyr Ile Glu Asp Phe Arg His 195 200 205 Val Glu Ile Gin Val Leu Ala Asp Asn Tyr Gly Asn Thr Ile His Leu

210 215 220

Gly Glu Arg Asp Cys Ser Ile Gin Arg Arg Leu Gin Lys Leu Leu Glu 225 230 235 240

Glu Ser Pro Ser Pro Ala Leu Asp Ser Glu Ile Arg Glu Gin Met Gly 245 250 255

Asp Ala Ala Val Lys Ala Ala Lys Ala Val Gly Tyr Thr Gly Ala Gly

260 265 270

Thr Val Glu Phe Ile Tyr Asp Tyr Asn Glu Gin Arg Tyr Tyr Phe Met 275 280 285

Glu Met Asn Thr Arg Ile Gin Val Glu His Pro Val Thr Glu Met Val

290 295 300

Thr Gly Thr Asp Leu Ile Lys Glu Gin Ile Lys Val Ala Ser Gly Met 305 310 315 320

Glu Leu Ser Leu Lys Gin Glu Asp Val Glu Phe Glu Gly Trp Ala Ile 325 330 335

Glu Cys Arg Ile Asn Ala Glu Asn Pro Ser Lys Asn Phe Met Pro Ser

340 345 350

Pro Gly Glu Ile Lys Met Tyr Leu Pro Pro Gly Gly Leu Gly Val Arg 355 360 365 Val Asp Ser Ala Ala Tyr Pro Gly Tyr Ser Ile Pro Pro Tyr Tyr Asp

370 375 380

Ser Met Ile Ala Lys Val Ile Thr Tyr Gly Lys Thr Arg Asp Glu Ala 385 390 395 400 Ile Ala Arg Met Lys Arg Ala Leu Ser Glu Phe Val Ile Glu Gly Ile 405 410 415

Glu Thr Thr Ile Pro Phe His Leu Lys Leu Leu Glu His Glu Thr Phe 420 425 430 Val Ser Gly Glu Phe Asn Thr Lys Phe Leu Glu Thr Tyr Asp Val Met 435 440 445

Gly Ser

450

<210> 179

<211> 408

<212> DNA

<213> Bacillus

<220>

<221> CDS

<222> (1) . . (408)

<223> Essential gene yghY (in accC operon)

<400> 179 atg aaa gac aac agc ttg ctt aaa atg gat cac gaa gat acg cat ttg 48

Met Lys Asp Asn Ser Leu Leu Lys Met Asp His Glu Asp Thr His Leu

1 5 10 15 ggt aag gtt gag att gca ccg gag gtc att gag gtc att gcc ggc att 96

Gly Lys Val Glu Ile Ala Pro Glu Val Ile Glu Val Ile Ala Gly Ile

20 25 30 gca gca tca gaa gtt gat gga gtt gcc gaa atg cgc gga aac ttt gcg 144

Ala Ala Ser Glu Val Asp Gly Val Ala Glu Met Arg Gly Asn Phe Ala

35 40 45 aca ggc gtc gtt gag cgc ttt ggc aaa gtg aat cat ggc aaa ggt gtc 192

Thr Gly Val Val Glu Arg Phe Gly Lys Val Asn His Gly Lys Gly Val

50 55 60 aaa gtc gac ctg gcg gat gac gga att acg atc gat gta tac tgt gtc 240

Lys Val Asp Leu Ala Asp Asp Gly Ile Thr Ile Asp Val Tyr Cys Val

65 70 75 80 gtt aca ttt ggc gtt tcg att ccg aaa gtt gca gca tcc gtt cag gaa 288 Val Thr Phe Gly Val Ser Ile Pro Lys Val Ala Ala Ser Val Gin Glu

85 90 95 aac att cgt caa acc tta tta aat atg acg tct ctt tcg att aac gag 336

Asn Ile Arg Gin Thr Leu Leu Asn Met Thr Ser Leu Ser Ile Asn Glu

100 105 110 atc aat att cac atc gtc ggc att caa ttt gac aca aaa gcc caa gaa 384 Ile Asn Ile His Ile Val Gly Ile Gin Phe Asp Thr Lys Ala Gin Glu

115 120 125 gtc caa atc gac gaa gaa atg taa 408 Val Gin Ile Asp Glu Glu Met

130 135 <210> 180

<211> 135

<212> PRT

<213> Bacillus

<400> 180

Met Lys Asp Asn Ser Leu Leu Lys Met Asp His Glu Asp Thr His Leu

1 5 10 15

Gly Lys Val Glu Ile Ala Pro Glu Val Ile Glu Val Ile Ala Gly Ile 20 25 30

Ala Ala Ser Glu Val Asp Gly Val Ala Glu Met Arg Gly Asn Phe Ala 35 40 45

Thr Gly Val Val Glu Arg Phe Gly Lys Val Asn His Gly Lys Gly Val 50 55 60

Lys Val Asp Leu Ala Asp Asp Gly Ile Thr Ile Asp Val Tyr Cys Val 65 70 75 80 Val Thr Phe Gly Val Ser Ile Pro Lys Val Ala Ala Ser Val Gin Glu 85 90 95

Asn Ile Arg Gin Thr Leu Leu Asn Met Thr Ser Leu Ser Ile Asn Glu 100 105 110 Ile Asn Ile His Ile Val Gly Ile Gin Phe Asp Thr Lys Ala Gin Glu

115 120 125 Val Gin Ile Asp Glu Glu Met

130 135

<210> 181

<211> 1344

<212> DNA

<213> Corynebacterium

<220>

<221> CDS

<222> (1) . . (1344 )

<223> Corynebacterium essential gene glmM

<400> 181 atg act cga ctt ttt gga act gat ggc gtc cgc gga cta gcc aat gaa 48

Met Thr Arg Leu Phe Gly Thr Asp Gly Val Arg Gly Leu Ala Asn Glu

1 5 10 15 gta ctc acc gca cct ttg gcc ctg aaa ctg ggt gcc gct gca gct cac 96 Val Leu Thr Ala Pro Leu Ala Leu Lys Leu Gly Ala Ala Ala Ala His

20 25 30 gta ctt acc gca gag aaa cgc gta gat gga cgc cgc ccg gtt gcg atc 144 Val Leu Thr Ala Glu Lys Arg Val Asp Gly Arg Arg Pro Val Ala Ile

35 40 45 gtt ggt agg gat cct cga gtc tct gga gag atg ctc gcg gca gca ctt 192 Val Gly Arg Asp Pro Arg Val Ser Gly Glu Met Leu Ala Ala Ala Leu

50 55 60 tca gca ggc atg gcc agc cag ggt gtt gat gtc att cgt gtt ggt gtc 240

Ser Ala Gly Met Ala Ser Gin Gly Val Asp Val Ile Arg Val Gly Val

65 70 75 80 atc cca acc cca gct gtt gca ttc ctc acc gat gat tat ggc gct gat 288 Ile Pro Thr Pro Ala Val Ala Phe Leu Thr Asp Asp Tyr Gly Ala Asp

85 90 95 atg ggc gtg atg att tct gca tcc cac aac cca atg ccg gac aac gga 336

Met Gly Val Met Ile Ser Ala Ser His Asn Pro Met Pro Asp Asn Gly

100 105 110 atc aag ttc ttc tcc gca ggt gga cac aag ctt cca gac cat gtg gaa 384 Ile Lys Phe Phe Ser Ala Gly Gly His Lys Leu Pro Asp His Val Glu

115 120 125 gac gag att gag cgt gtc atg gac agc ttg cct gcc gaa ggc cca acc 432

Asp Glu Ile Glu Arg Val Met Asp Ser Leu Pro Ala Glu Gly Pro Thr

130 135 140 ggg cat gga gtt ggc cgt gtc att gaa gaa gca acc gat gca caa gac 480

Gly His Gly Val Gly Arg Val Ile Glu Glu Ala Thr Asp Ala Gin Asp

145 150 155 160 cgc tac ctg gag cac ctg aag gaa gct gtt cct acg tca ctt gaa ggc 528

Arg Tyr Leu Glu His Leu Lys Glu Ala Val Pro Thr Ser Leu Glu Gly

165 170 175 atc aag att gtt gtg gat gca gcc aat ggt gca gca agc gtt gta gct 576 Ile Lys Ile Val Val Asp Ala Ala Asn Gly Ala Ala Ser Val Val Ala

180 185 190 cca aag gct tat gaa gca gca ggt gca act gta att gct att cat aac 624

Pro Lys Ala Tyr Glu Ala Ala Gly Ala Thr Val Ile Ala Ile His Asn

195 200 205 aag cca gac tcc tac aac atc aac atg gat tgc ggt tcc acc cac att 672

Lys Pro Asp Ser Tyr Asn Ile Asn Met Asp Cys Gly Ser Thr His Ile

210 215 220 gac cag gtg cag gcg gca gtt ctg aag cac ggt gct gac ctt gga ctc 720

Asp Gin Val Gin Ala Ala Val Leu Lys His Gly Ala Asp Leu Gly Leu

225 230 235 240 gca cac gac ggc gat gct gac cgt tgt ttg gct gtg gac aaa gat gga 768

Ala His Asp Gly Asp Ala Asp Arg Cys Leu Ala Val Asp Lys Asp Gly

245 250 255 aac ctc att gac ggt gat cag atc atg gca atg tta gcc att gcg atg 816

Asn Leu Ile Asp Gly Asp Gin Ile Met Ala Met Leu Ala Ile Ala Met

260 265 270 aaa gaa aac ggc gag ctg cgc aag aac acc ctc gtg ggc acc gtc atg 864

Lys Glu Asn Gly Glu Leu Arg Lys Asn Thr Leu Val Gly Thr Val Met 275 280 285 agc aac ttg gga ctg aag att gcc atg gat gaa gcc gga att acg ctg 912

Ser Asn Leu Gly Leu Lys Ile Ala Met Asp Glu Ala Gly Ile Thr Leu

290 295 300 cga acc acc aag gtg ggc gac cgc tac gtg ctg gaa gac ctc aat gcg 960

Arg Thr Thr Lys Val Gly Asp Arg Tyr Val Leu Glu Asp Leu Asn Ala

305 310 315 320 ggt gga ttc agc ctt ggt ggc gag cag tct gga cac att gtt ctt cca 1008

Gly Gly Phe Ser Leu Gly Gly Glu Gin Ser Gly His Ile Val Leu Pro

325 330 335 gat cac ggc acc act ggc gat gga act ttg act ggt ctt tec atc atg 1056

Asp His Gly Thr Thr Gly Asp Gly Thr Leu Thr Gly Leu Ser Ile Met

340 345 350 gcg cgc atg gct gaa acc gga aaa tcc ttg ggc gag ttg gca caa gcg 1104

Ala Arg Met Ala Glu Thr Gly Lys Ser Leu Gly Glu Leu Ala Gin Ala

355 360 365 atg acg gtt ctg cca cag gtt ctg atc aat gtg cca gtc tct gat aaa 1152

Met Thr Val Leu Pro Gin Val Leu Ile Asn Val Pro Val Ser Asp Lys

370 375 380 tcc acc atc gtt tct cac cca agt gtc gtt gca gca atc gct gaa gct 1200

Ser Thr Ile Val Ser His Pro Ser Val Val Ala Ala Ile Ala Glu Ala

385 390 395 400 gaa gcg gag ttg ggt tct acc ggt cgc gtg ctt ctt cgc gct tct ggc 1248

Glu Ala Glu Leu Gly Ser Thr Gly Arg Val Leu Leu Arg Ala Ser Gly

405 410 415 acc gag gag ctt ttc cgt gtg atg gtc gaa gct ggc gat aag gaa caa 1296

Thr Glu Glu Leu Phe Arg Val Met Val Glu Ala Gly Asp Lys Glu Gin

420 425 430 gca cgc cgc att gcg gga cgt ctt tct gcc gtg gtc gcg gaa gtc taa 1344

Ala Arg Arg Ile Ala Gly Arg Leu Ser Ala Val Val Ala Glu Val

435 440 445

<210> 182

<211> 447

<212> PRT

<213> Corynebacterium

<400> 182

Met Thr Arg Leu Phe Gly Thr Asp Gly Val Arg Gly Leu Ala Asn Glu

1 5 10 15 Val Leu Thr Ala Pro Leu Ala Leu Lys Leu Gly Ala Ala Ala Ala His 20 25 30 Val Leu Thr Ala Glu Lys Arg Val Asp Gly Arg Arg Pro Val Ala Ile 35 40 45 Val Gly Arg Asp Pro Arg Val Ser Gly Glu Met Leu Ala Ala Ala Leu 50 55 60

Ser Ala Gly Met Ala Ser Gin Gly Val Asp Val Ile Arg Val Gly Val 65 70 75 80 Ile Pro Thr Pro Ala Val Ala Phe Leu Thr Asp Asp Tyr Gly Ala Asp 85 90 95

Met Gly Val Met Ile Ser Ala Ser His Asn Pro Met Pro Asp Asn Gly

100 105 110 Ile Lys Phe Phe Ser Ala Gly Gly His Lys Leu Pro Asp His Val Glu

115 120 125

Asp Glu Ile Glu Arg Val Met Asp Ser Leu Pro Ala Glu Gly Pro Thr

130 135 140

Gly His Gly Val Gly Arg Val Ile Glu Glu Ala Thr Asp Ala Gin Asp 145 150 155 160

Arg Tyr Leu Glu His Leu Lys Glu Ala Val Pro Thr Ser Leu Glu Gly 165 170 175 Ile Lys Ile Val Val Asp Ala Ala Asn Gly Ala Ala Ser Val Val Ala

180 185 190

Pro Lys Ala Tyr Glu Ala Ala Gly Ala Thr Val Ile Ala Ile His Asn 195 200 205

Lys Pro Asp Ser Tyr Asn Ile Asn Met Asp Cys Gly Ser Thr His Ile

210 215 220

Asp Gin Val Gin Ala Ala Val Leu Lys His Gly Ala Asp Leu Gly Leu 225 230 235 240

Ala His Asp Gly Asp Ala Asp Arg Cys Leu Ala Val Asp Lys Asp Gly 245 250 255

Asn Leu Ile Asp Gly Asp Gin Ile Met Ala Met Leu Ala Ile Ala Met

260 265 270

Lys Glu Asn Gly Glu Leu Arg Lys Asn Thr Leu Val Gly Thr Val Met 275 280 285

Ser Asn Leu Gly Leu Lys Ile Ala Met Asp Glu Ala Gly Ile Thr Leu

290 295 300 Arg Thr Thr Lys Val Gly Asp Arg Tyr Val Leu Glu Asp Leu Asn Ala 305 310 315 320

Gly Gly Phe Ser Leu Gly Gly Glu Gin Ser Gly His Ile Val Leu Pro 325 330 335

Asp His Gly Thr Thr Gly Asp Gly Thr Leu Thr Gly Leu Ser Ile Met 340 345 350

Ala Arg Met Ala Glu Thr Gly Lys Ser Leu Gly Glu Leu Ala Gin Ala 355 360 365

Met Thr Val Leu Pro Gin Val Leu Ile Asn Val Pro Val Ser Asp Lys 370 375 380

Ser Thr Ile Val Ser His Pro Ser Val Val Ala Ala Ile Ala Glu Ala

385 390 395 400

Glu Ala Glu Leu Gly Ser Thr Gly Arg Val Leu Leu Arg Ala Ser Gly

405 410 415

Thr Glu Glu Leu Phe Arg Val Met Val Glu Ala Gly Asp Lys Glu Gin

420 425 430

Ala Arg Arg Ile Ala Gly Arg Leu Ser Ala Val Val Ala Glu Val 435 440 445

<210> 183

<211> 1086

<212> DNA

<213> Corynebacterium

<220>

<221> CDS

<222> (1) . . (1086)

<223> Corynebacterium essential gene air

<400> 183 atg aac ttg ctg acc acc aaa att gac ctg gat gcc atc gcc cat aac 48

Met Asn Leu Leu Thr Thr Lys Ile Asp Leu Asp Ala Ile Ala His Asn

1 5 10 15 acg agg gtg ctt aaa caa atg gcg ggt ccg gcg aag ctg atg gcg gtg 96

Thr Arg Val Leu Lys Gin Met Ala Gly Pro Ala Lys Leu Met Ala Val

20 25 30 gtg aag gcg aat gca tat aac cat ggc gtg gag aag gtc gct ccg gtt 144 Val Lys Ala Asn Ala Tyr Asn His Gly Val Glu Lys Val Ala Pro Val

35 40 45 att gct gcg cat ggt gcg gat gcg ttt ggt gtg gca act ctt gcg gag 192 Ile Ala Ala His Gly Ala Asp Ala Phe Gly Val Ala Thr Leu Ala Glu

50 55 60 gct atg cag ttg cgt gat atc ggc atc agc caa gag gtt ttg tgt tgg 240

Ala Met Gin Leu Arg Asp Ile Gly Ile Ser Gin Glu Val Leu Cys Trp

65 70 75 80 att tgg aca ccg gag cag gat ttc cgc gcc gcc att gat cgc aat att 288 Ile Trp Thr Pro Glu Gin Asp Phe Arg Ala Ala Ile Asp Arg Asn Ile

85 90 95 gat ttg gct gtt att tct ccc gcg cat gcc aaa gcc ttg atc gac act 336

Asp Leu Ala Val Ile Ser Pro Ala His Ala Lys Ala Leu Ile Asp Thr

100 105 110 gat gcg gag cat att egg gtg tcc atc aag att gat tct ggg ttg cat 384

Asp Ala Glu His Ile Arg Val Ser Ile Lys Ile Asp Ser Gly Leu His

115 120 125 cgt tcg ggt gtg gat gag cag gag tgg gag ggc gtg ttc agc gcg ttg 432

Arg Ser Gly Val Asp Glu Gin Glu Trp Glu Gly Val Phe Ser Ala Leu

130 135 140 gct gct gcc ccg cac att gag gtc acg ggc atg ttc acg cac ttg gcg 480

Ala Ala Ala Pro His Ile Glu Val Thr Gly Met Phe Thr His Leu Ala

145 150 155 160 tgc gcg gat gag cca gag aat ccg gaa act gat cgc caa att att gct 528

Cys Ala Asp Glu Pro Glu Asn Pro Glu Thr Asp Arg Gin Ile Ile Ala

165 170 175 ttt cga cgc gcc ctt gcg ctc gcc cgc aag cac ggg ctt gag tgc ccg 576

Phe Arg Arg Ala Leu Ala Leu Ala Arg Lys His Gly Leu Glu Cys Pro

180 185 190 gtc aac cac gta tgc aac tca cct gca ttt ttg act cga tct gat tta 624 Val Asn His Val Cys Asn Ser Pro Ala Phe Leu Thr Arg Ser Asp Leu

195 200 205 cac atg gag atg gtc cga ccg ggt ttg gcc ttt tat ggg ttg gaa ccc 672

His Met Glu Met Val Arg Pro Gly Leu Ala Phe Tyr Gly Leu Glu Pro

210 215 220 gtg gcg gga cga gag cat ggt ttg aag ccg gcg atg acg tgg gag gcg 720 Val Ala Gly Arg Glu His Gly Leu Lys Pro Ala Met Thr Trp Glu Ala

225 230 235 240 aag gtg agc gtc gta aag caa att gaa gct gga caa ggc acc tct tat 768

Lys Val Ser Val Val Lys Gin Ile Glu Ala Gly Gin Gly Thr Ser Tyr

245 250 255 ggc ctg acc tgg cgc gct gag gat cgc ggc ttt gtg gct gtg gtg cct 816

Gly Leu Thr Trp Arg Ala Glu Asp Arg Gly Phe Val Ala Val Val Pro

260 265 270 gcg ggc tat gcc gat ggc atg ccg egg cat gcc cag ggg aaa ttc tcc 864

Ala Gly Tyr Ala Asp Gly Met Pro Arg His Ala Gin Gly Lys Phe Ser

275 280 285 gtc acg att gat ggc gtg gac tat ccg cag gtt gga cgc gtg tgc atg 912 Val Thr Ile Asp Gly Val Asp Tyr Pro Gin Val Gly Arg Val Cys Met

290 295 300 gat cag ttc gtt att tct ttg ggc gac aat ccg cac ggt gtg gaa gct 960

Asp Gin Phe Val Ile Ser Leu Gly Asp Asn Pro His Gly Val Glu Ala 305 310 315 320 ggc gcg aag gcc gtg ata ttc ggt gag aat ggg cat gac gca act gat 1008 Gly Ala Lys Ala Val Ile Phe Gly Glu Asn Gly His Asp Ala Thr Asp 325 330 335 ttt gcg gag cgt tta gac acc att aac tat gag gta gtg tgc cga cca 1056

Phe Ala Glu Arg Leu Asp Thr Ile Asn Tyr Glu Val Val Cys Arg Pro 340 345 350 acc ggc cga act gtc cgc gca tat gtt taa 1086 Thr Gly Arg Thr Val Arg Ala Tyr Val 355 360

<210> 184

<211> 361

<212> PRT

<213> Corynebacterium

<400> 184

Met Asn Leu Leu Thr Thr Lys Ile Asp Leu Asp Ala Ile Ala His Asn

1 5 10 15

Thr Arg Val Leu Lys Gin Met Ala Gly Pro Ala Lys Leu Met Ala Val 20 25 30 Val Lys Ala Asn Ala Tyr Asn His Gly Val Glu Lys Val Ala Pro Val 35 40 45 Ile Ala Ala His Gly Ala Asp Ala Phe Gly Val Ala Thr Leu Ala Glu

50 55 60

Ala Met Gin Leu Arg Asp Ile Gly Ile Ser Gin Glu Val Leu Cys Trp 65 70 75 80 Ile Trp Thr Pro Glu Gin Asp Phe Arg Ala Ala Ile Asp Arg Asn Ile

85 90 95

Asp Leu Ala Val Ile Ser Pro Ala His Ala Lys Ala Leu Ile Asp Thr 100 105 110

Asp Ala Glu His Ile Arg Val Ser Ile Lys Ile Asp Ser Gly Leu His 115 120 125

Arg Ser Gly Val Asp Glu Gin Glu Trp Glu Gly Val Phe Ser Ala Leu

130 135 140

Ala Ala Ala Pro His Ile Glu Val Thr Gly Met Phe Thr His Leu Ala 145 150 155 160

Cys Ala Asp Glu Pro Glu Asn Pro Glu Thr Asp Arg Gin Ile Ile Ala 165 170 175

Phe Arg Arg Ala Leu Ala Leu Ala Arg Lys His Gly Leu Glu Cys Pro 180 185 190 Val Asn His Val Cys Asn Ser Pro Ala Phe Leu Thr Arg Ser Asp Leu 195 200 205

His Met Glu Met Val Arg Pro Gly Leu Ala Phe Tyr Gly Leu Glu Pro

210 215 220 Val Ala Gly Arg Glu His Gly Leu Lys Pro Ala Met Thr Trp Glu Ala 225 230 235 240

Lys Val Ser Val Val Lys Gin Ile Glu Ala Gly Gin Gly Thr Ser Tyr

245 250 255

Gly Leu Thr Trp Arg Ala Glu Asp Arg Gly Phe Val Ala Val Val Pro

260 265 270

Ala Gly Tyr Ala Asp Gly Met Pro Arg His Ala Gin Gly Lys Phe Ser 275 280 285 Val Thr Ile Asp Gly Val Asp Tyr Pro Gin Val Gly Arg Val Cys Met

290 295 300

Asp Gin Phe Val Ile Ser Leu Gly Asp Asn Pro His Gly Val Glu Ala 305 310 315 320

Gly Ala Lys Ala Val Ile Phe Gly Glu Asn Gly His Asp Ala Thr Asp

325 330 335

Phe Ala Glu Arg Leu Asp Thr Ile Asn Tyr Glu Val Val Cys Arg Pro

340 345 350

Thr Gly Arg Thr Val Arg Ala Tyr Val 355 360

<210> 185

<211> 1119

<212> DNA

<213> Aspergillus oryzae

<220>

<221> CDS

<222> (1) . . (1119)

<223> Aspergillus essential gene ARG3

<400> 185 atg act tgc ggt ctg aag ctg gcc gcc gct cgt tac ggc aac cat acc 48 Met Thr Cys Gly Leu Lys Leu Ala Ala Ala Arg Tyr Gly Asn His Thr 1 5 10 15 ttg cgc cag aag att cca ctc aac gct gtg cgc cga tac act tct cac 96

Leu Arg Gin Lys Ile Pro Leu Asn Ala Val Arg Arg Tyr Thr Ser His

20 25 30 act gct act tct acg act ccc ccc acc tcg cca ttc gct cct cgc cat 144

Thr Ala Thr Ser Thr Thr Pro Pro Thr Ser Pro Phe Ala Pro Arg His

35 40 45 ttt ctt tec atc gct gat ctt acc tca acg gag ttc gca acc ctc gtc 192

Phe Leu Ser Ile Ala Asp Leu Thr Ser Thr Glu Phe Ala Thr Leu Val

50 55 60 cgt aat gca tct tca cac aaa cga acc atc aag tcg gga tca atc cct 240

Arg Asn Ala Ser Ser His Lys Arg Thr Ile Lys Ser Gly Ser Ile Pro

65 70 75 80 cag aac ctg ctt ggt tct atg acc gga cag act gta gct atg ctg ttc 288

Gin Asn Leu Leu Gly Ser Met Thr Gly Gin Thr Val Ala Met Leu Phe

85 90 95 agc aaa cgc agt acc aga acc agg ata tct act gaa ggg gct gtg gtg 336

Ser Lys Arg Ser Thr Arg Thr Arg Ile Ser Thr Glu Gly Ala Val Val

100 105 110 cgc ttg gga gga cat cca atg ttt ctg ggc aag gat gat att caa ctc 384

Arg Leu Gly Gly His Pro Met Phe Leu Gly Lys Asp Asp Ile Gin Leu

115 120 125 ggt gtc aat gag tcc ttg tat gac tct gcg gtt gta atc tcc tcc atg 432

Gly Val Asn Glu Ser Leu Tyr Asp Ser Ala Val Val Ile Ser Ser Met

130 135 140 gtc tct tgt att gta gcc cga gtc ggc aag cac gct gag gtt gca gac 480 Val Ser Cys Ile Val Ala Arg Val Gly Lys His Ala Glu Val Ala Asp

145 150 155 160 ctt gcc aag cat tca acc gtg cct gtt atc aac gct ctc tgt gac tcc 528

Leu Ala Lys His Ser Thr Val Pro Val Ile Asn Ala Leu Cys Asp Ser

165 170 175 ttc cac cct ctc cag gcc att gcc gat ttt cag acc atc tac gaa aca 576

Phe His Pro Leu Gin Ala Ile Ala Asp Phe Gin Thr Ile Tyr Glu Thr

180 185 190 ttt aca cct aag gcc cat cgc tct gac agt ttg ggt ctg gag ggc ctc 624

Phe Thr Pro Lys Ala His Arg Ser Asp Ser Leu Gly Leu Glu Gly Leu

195 200 205 aag att gcc tgg gtc ggt gat gca aac aac gtt ctg ttc gac atg gca 672

Lys Ile Ala Trp Val Gly Asp Ala Asn Asn Val Leu Phe Asp Met Ala

210 215 220 att gct gct acc aag atg ggt att gat atc gcc gtt gcg act ccg aag 720 Ile Ala Ala Thr Lys Met Gly Ile Asp Ile Ala Val Ala Thr Pro Lys

225 230 235 240 ggc tat gag att cct gcc ccg atg ttg gag ctc atc aag cag gcc agc 768

Gly Tyr Glu Ile Pro Ala Pro Met Leu Glu Leu Ile Lys Gin Ala Ser

245 250 255 aac ggt gtc tca aag cca gga aag att att gag acc aac gtt cct gag 816

Asn Gly Val Ser Lys Pro Gly Lys Ile Ile Glu Thr Asn Val Pro Glu

260 265 270 gag gcg gtt aag ggt gcg gat atc ctg gta act gac acc tgg gtt tcc 864

Glu Ala Val Lys Gly Ala Asp Ile Leu Val Thr Asp Thr Trp Val Ser

275 280 285 atg gga cag gag gca gag tcg atc aag egg gtg aag gac ttc gag ggc 912

Met Gly Gin Glu Ala Glu Ser Ile Lys Arg Val Lys Asp Phe Glu Gly

290 295 300 ttc cag atc act tcg gaa ctt gcc aag cgc ggt ggt gcg aac gaa ggc 960

Phe Gin Ile Thr Ser Glu Leu Ala Lys Arg Gly Gly Ala Asn Glu Gly

305 310 315 320 tgg aag ttc atg cac tgc ctg cct cgt cac cct gag gaa gtc agc gat 1008

Trp Lys Phe Met His Cys Leu Pro Arg His Pro Glu Glu Val Ser Asp

325 330 335 gaa gtt ttc tac agc cca egg tcc ttg gtc ttc ccc gaa gcg gag aat 1056

Glu Val Phe Tyr Ser Pro Arg Ser Leu Val Phe Pro Glu Ala Glu Asn

340 345 350 egg ttg tgg gct gcg atc tcg gcc atg gag ggc ttt gtt gtc aat aag 1104

Arg Leu Trp Ala Ala Ile Ser Ala Met Glu Gly Phe Val Val Asn Lys

355 360 365 gga agg att gag taa 1119

Gly Arg Ile Glu 370

<210> 186

<211> 372

<212> PRT

<213> Aspergillus oryzae

<400> 186

Met Thr Cys Gly Leu Lys Leu Ala Ala Ala Arg Tyr Gly Asn His Thr 1 5 10 15

Leu Arg Gin Lys Ile Pro Leu Asn Ala Val Arg Arg Tyr Thr Ser His 20 25 30

Thr Ala Thr Ser Thr Thr Pro Pro Thr Ser Pro Phe Ala Pro Arg His 35 40 45

Phe Leu Ser Ile Ala Asp Leu Thr Ser Thr Glu Phe Ala Thr Leu Val 50 55 60

Arg Asn Ala Ser Ser His Lys Arg Thr Ile Lys Ser Gly Ser Ile Pro 65 70 75 80

Gin Asn Leu Leu Gly Ser Met Thr Gly Gin Thr Val Ala Met Leu Phe 85 90 95 Ser Lys Arg Ser Thr Arg Thr Arg Ile Ser Thr Glu Gly Ala Val Val

100 105 110

Arg Leu Gly Gly His Pro Met Phe Leu Gly Lys Asp Asp Ile Gin Leu 115 120 125

Gly Val Asn Glu Ser Leu Tyr Asp Ser Ala Val Val Ile Ser Ser Met

130 135 140 Val Ser Cys Ile Val Ala Arg Val Gly Lys His Ala Glu Val Ala Asp 145 150 155 160

Leu Ala Lys His Ser Thr Val Pro Val Ile Asn Ala Leu Cys Asp Ser 165 170 175

Phe His Pro Leu Gin Ala Ile Ala Asp Phe Gin Thr Ile Tyr Glu Thr

180 185 190

Phe Thr Pro Lys Ala His Arg Ser Asp Ser Leu Gly Leu Glu Gly Leu 195 200 205

Lys Ile Ala Trp Val Gly Asp Ala Asn Asn Val Leu Phe Asp Met Ala

210 215 220 Ile Ala Ala Thr Lys Met Gly Ile Asp Ile Ala Val Ala Thr Pro Lys

225 230 235 240

Gly Tyr Glu Ile Pro Ala Pro Met Leu Glu Leu Ile Lys Gin Ala Ser 245 250 255

Asn Gly Val Ser Lys Pro Gly Lys Ile Ile Glu Thr Asn Val Pro Glu

260 265 270

Glu Ala Val Lys Gly Ala Asp Ile Leu Val Thr Asp Thr Trp Val Ser 275 280 285

Met Gly Gin Glu Ala Glu Ser Ile Lys Arg Val Lys Asp Phe Glu Gly

290 295 300

Phe Gin Ile Thr Ser Glu Leu Ala Lys Arg Gly Gly Ala Asn Glu Gly 305 310 315 320

Trp Lys Phe Met His Cys Leu Pro Arg His Pro Glu Glu Val Ser Asp 325 330 335

Glu Val Phe Tyr Ser Pro Arg Ser Leu Val Phe Pro Glu Ala Glu Asn 340 345 350

Arg Leu Trp Ala Ala Ile Ser Ala Met Glu Gly Phe Val Val Asn Lys 355 360 365

Gly Arg Ile Glu 370

<210> 187

<211> 2184

<212> DNA

<213> Aspergillus oryzae

<220>

<221> CDS

<222> (1) . . (2184 )

<223> Aspergillus essential gene adeA

<400> 187 atg ggt gct aga ctc aca tat ata aat agt atc att ctc cta tcc gat Met Gly Ala Arg Leu Thr Tyr Ile Asn Ser Ile Ile Leu Leu Ser Asp 1 5 10 15 tgt acc atc gca cat aat ctt gtc tca ttc cac gat ttg aat gcg ggg 96

Cys Thr Ile Ala His Asn Leu Val Ser Phe His Asp Leu Asn Ala Gly

20 25 30 ttc aga acc aac att cat tta cgt ctc ccc aaa caa aca tcc gac ttc 144

Phe Arg Thr Asn Ile His Leu Arg Leu Pro Lys Gin Thr Ser Asp Phe

35 40 45 cac acc atg tct tcc tct ttg agc acg ggt tcc ttc caa acc ctc act 192

His Thr Met Ser Ser Ser Leu Ser Thr Gly Ser Phe Gin Thr Leu Thr

50 55 60 ttt cac cct gac aac acg gtg att ata cag gat aaa atc tac ggg gag 240

Phe His Pro Asp Asn Thr Val Ile Ile Gin Asp Lys Ile Tyr Gly Glu

65 70 75 80 cac acc atc tca gag ccg gtt cta gcc gag ctg ctt egg tgt cca gct 288

His Thr Ile Ser Glu Pro Val Leu Ala Glu Leu Leu Arg Cys Pro Ala

85 90 95 cta ctt cga ctc gcg ggc atc ggt ctt cac ggt caa acc gat ctt ctg 336

Leu Leu Arg Leu Ala Gly Ile Gly Leu His Gly Gin Thr Asp Leu Leu

100 105 110 ggt ata acc cat aca gtg act cga cta gag cac tcc ata ggc gcg tcg 384

Gly Ile Thr His Thr Val Thr Arg Leu Glu His Ser Ile Gly Ala Ser

115 120 125 cta tta gtg cgc aaa gtc ggc ggt agc atc gga gag caa gtc gca ggt 432

Leu Leu Val Arg Lys Val Gly Gly Ser Ile Gly Glu Gin Val Ala Gly

130 135 140 ctt ctg cac gac atc agc cac acg gta ctc agc cat gac gtc gat ggg 480

Leu Leu His Asp Ile Ser His Thr Val Leu Ser His Asp Val Asp Gly

145 150 155 160 gcc ttg tcg aag cca ggg gag agc tat cat gag gtc caa aag tca egg 528 Ala Leu Ser Lys Pro Gly Glu Ser Tyr His Glu Val Gin Lys Ser Arg 165 170 175 tat atc atg acc act gag ttg cca cga ata ctc acc aaa cat ggc ttt 576

Tyr Ile Met Thr Thr Glu Leu Pro Arg Ile Leu Thr Lys His Gly Phe

180 185 190 gtt gat cta aag ccg ttt gat gaa gag ctt tat cct ctg gtg gag aga 624 Val Asp Leu Lys Pro Phe Asp Glu Glu Leu Tyr Pro Leu Val Glu Arg

195 200 205 ccc gct cct cat ctc tgc gct gat cgt ctc gat tac tct cta cga gat 672

Pro Ala Pro His Leu Cys Ala Asp Arg Leu Asp Tyr Ser Leu Arg Asp

210 215 220 gct gtc gcg ttc ggt aaa ctt gcg ata gag gat gcg cgc cgc gtg tat 720

Ala Val Ala Phe Gly Lys Leu Ala Ile Glu Asp Ala Arg Arg Val Tyr

225 230 235 240 gac tca ttg aca gca ttc ccg gac gcg tct tca cca cat cgc ttg ctg 768

Asp Ser Leu Thr Ala Phe Pro Asp Ala Ser Ser Pro His Arg Leu Leu

245 250 255 gtt ctt cga gac att gat cta gcc tta gct tat gct agg gcc tat ggt 816 Val Leu Arg Asp Ile Asp Leu Ala Leu Ala Tyr Ala Arg Ala Tyr Gly

260 265 270 gaa tgc gat agg gat gtg tgg tgc aat ccg gct cat gcc gtc atg tcc 864

Glu Cys Asp Arg Asp Val Trp Cys Asn Pro Ala His Ala Val Met Ser

275 280 285 agg aag ata ggt cag ttg att ggg gat cta gtg caa caa gga tcg ttg 912

Arg Lys Ile Gly Gin Leu Ile Gly Asp Leu Val Gin Gin Gly Ser Leu

290 295 300 aag gaa gag gtc cta tgg aat cta tcg gac cgc gaa ttt tgg gag ctc 960

Lys Glu Glu Val Leu Trp Asn Leu Ser Asp Arg Glu Phe Trp Glu Leu

305 310 315 320 ctg aaa agt aaa gtt gac tct aaa gga ttg gag acg att aaa cac atc 1008

Leu Lys Ser Lys Val Asp Ser Lys Gly Leu Glu Thr Ile Lys His Ile

325 330 335 gaa gca ggg ccg cat gcg gaa gat tat cat cgt cta cct cgt ggt acc 1056

Glu Ala Gly Pro His Ala Glu Asp Tyr His Arg Leu Pro Arg Gly Thr

340 345 350 aag att cgc aca atc gat cct gat ctg ctc ctc ccg ggt gct ggg caa 1104

Lys Ile Arg Thr Ile Asp Pro Asp Leu Leu Leu Pro Gly Ala Gly Gin

355 360 365 ccc tcc cca ctg tct ttt gtg aaa cct gaa tgg gct aag gag aga caa 1152

Pro Ser Pro Leu Ser Phe Val Lys Pro Glu Trp Ala Lys Glu Arg Gin

370 375 380 gac ttc att cag gct cgc cag gcc tta cta gca tct ttt act gtc tac 1200

Asp Phe Ile Gin Ala Arg Gin Ala Leu Leu Ala Ser Phe Thr Val Tyr

385 390 395 400 atc aac ggc ggc gtg agc tgg gaa caa gat agt att cag cca caa ttc 1248 Ile Asn Gly Gly Val Ser Trp Glu Gin Asp Ser Ile Gin Pro Gin Phe

405 410 415 gag aac aat aat ccc atc atg tct gaa gct ctg aca aac acc gac ctt 1296 Glu Asn Asn Asn Pro Ile Met Ser Glu Ala Leu Thr Asn Thr Asp Leu 420 425 430 cag ggt gct ctc cct ctc atc gct cga ggc aag gtt cgc gac ctg tac 1344 Gin Gly Ala Leu Pro Leu Ile Ala Arg Gly Lys Val Arg Asp Leu Tyr 435 440 445 gat gtc gac gag aag acc ctc ctc ttc gtc gca acg gac cgc atc tcc 1392 Asp Val Asp Glu Lys Thr Leu Leu Phe Val Ala Thr Asp Arg Ile Ser 450 455 460 gcg tac gat gtg atc atg gag aat gga atc ccc gag aaa ggc atc ctc 1440 Ala Tyr Asp Val Ile Met Glu Asn Gly Ile Pro Glu Lys Gly Ile Leu 465 470 475 480 cta act cta tgc acg aaa acc tgg ttc aag atc ctc tcc gac aaa atc 1488 Leu Thr Leu Cys Thr Lys Thr Trp Phe Lys Ile Leu Ser Asp Lys Ile 485 490 495 ccc tct ctc cgc acc cac ttc ctc acc ctc gac ctc ccg cct cag atc 1536 Pro Ser Leu Arg Thr His Phe Leu Thr Leu Asp Leu Pro Pro Gin Ile 500 505 510 ccc gag tcg ttg cgt ccc gtc ctc cag aac aga agc atg cag gtg cgc 1584 Pro Glu Ser Leu Arg Pro Val Leu Gin Asn Arg Ser Met Gin Val Arg 515 520 525 aag ctg aag atc ctc ccc att gag gcc atc gtc cgc ggt tac atc acc 1632 Lys Leu Lys Ile Leu Pro Ile Glu Ala Ile Val Arg Gly Tyr Ile Thr 530 535 540 ggt tcc gcc tgg aac gag tac aag aaa tcc ggc acc gtg cac ggg atc 1680 Gly Ser Ala Trp Asn Glu Tyr Lys Lys Ser Gly Thr Val His Gly Ile 545 550 555 560 aag gtc gcc gag ggt ctg agg gag agc gag gcc ttc cct gat ggt cct 1728 Lys Val Ala Glu Gly Leu Arg Glu Ser Glu Ala Phe Pro Asp Gly Pro 565 570 575 att tac act ccc agt acc aag gcg gag cag ggc gag cat gat gag aac 1776 Ile Tyr Thr Pro Ser Thr Lys Ala Glu Gin Gly Glu His Asp Glu Asn 580 585 590 att cat ccg gat cag gct gtc gct att gtt ggt gaa cgt tat gcc tct 1824 Ile His Pro Asp Gin Ala Val Ala Ile Val Gly Glu Arg Tyr Ala Ser 595 600 605 aag atc gcc tct ctt gcg gtt cag ctt tac aag gtc gct cac gag tat 1872 Lys Ile Ala Ser Leu Ala Val Gin Leu Tyr Lys Val Ala His Glu Tyr 610 615 620 gcc ctt act cgt ggt gtg atc att gcc gac acc aag ttc gag ttc ggt 1920 Ala Leu Thr Arg Gly Val Ile Ile Ala Asp Thr Lys Phe Glu Phe Gly 625 630 635 640 ttg gat cct gag act gac gag gtt gtg ctg gcc gac gag gtc ctc acc 1968 Leu Asp Pro Glu Thr Asp Glu Val Val Leu Ala Asp Glu Val Leu Thr 645 650 655 ccc gat tca tcg cgt ttc tgg cct aag gac tcg tac gag atc ggt cgt 2016 Pro Asp Ser Ser Arg Phe Trp Pro Lys Asp Ser Tyr Glu Ile Gly Arg 660 665 670 gga cag cag agc ttc gat aag cag ttc ttg cgt gac tgg ttg acg agc 2064

Gly Gin Gin Ser Phe Asp Lys Gin Phe Leu Arg Asp Trp Leu Thr Ser

675 680 685 gag gga ctg aag ggc aag cct ggt gtg egg atg acc gat gag att gcg 2112

Glu Gly Leu Lys Gly Lys Pro Gly Val Arg Met Thr Asp Glu Ile Ala

690 695 700 cag aag acc agc gct aag tac egg gag gca tat gag egg atc act ggt 2160

Gin Lys Thr Ser Ala Lys Tyr Arg Glu Ala Tyr Glu Arg Ile Thr Gly

705 710 715 720 gag act aag gtt cct gcg gtc tag 2184

Glu Thr Lys Val Pro Ala Val 725

<210> 188

<211> 727

<212> PRT

<213> Aspergillus oryzae

<400> 188

Met Gly Ala Arg Leu Thr Tyr Ile Asn Ser Ile Ile Leu Leu Ser Asp

1 5 10 15

Cys Thr Ile Ala His Asn Leu Val Ser Phe His Asp Leu Asn Ala Gly 20 25 30

Phe Arg Thr Asn Ile His Leu Arg Leu Pro Lys Gin Thr Ser Asp Phe 35 40 45

His Thr Met Ser Ser Ser Leu Ser Thr Gly Ser Phe Gin Thr Leu Thr

50 55 60

Phe His Pro Asp Asn Thr Val Ile Ile Gin Asp Lys Ile Tyr Gly Glu 65 70 75 80

His Thr Ile Ser Glu Pro Val Leu Ala Glu Leu Leu Arg Cys Pro Ala 85 90 95

Leu Leu Arg Leu Ala Gly Ile Gly Leu His Gly Gin Thr Asp Leu Leu 100 105 110

Gly Ile Thr His Thr Val Thr Arg Leu Glu His Ser Ile Gly Ala Ser

115 120 125

Leu Leu Val Arg Lys Val Gly Gly Ser Ile Gly Glu Gin Val Ala Gly

130 135 140

Leu Leu His Asp Ile Ser His Thr Val Leu Ser His Asp Val Asp Gly 145 150 155 160

Ala Leu Ser Lys Pro Gly Glu Ser Tyr His Glu Val Gin Lys Ser Arg

165 170 175

Tyr Ile Met Thr Thr Glu Leu Pro Arg Ile Leu Thr Lys His Gly Phe 180 185 190 Val Asp Leu Lys Pro Phe Asp Glu Glu Leu Tyr Pro Leu Val Glu Arg 195 200 205

Pro Ala Pro His Leu Cys Ala Asp Arg Leu Asp Tyr Ser Leu Arg Asp

210 215 220

Ala Val Ala Phe Gly Lys Leu Ala Ile Glu Asp Ala Arg Arg Val Tyr 225 230 235 240

Asp Ser Leu Thr Ala Phe Pro Asp Ala Ser Ser Pro His Arg Leu Leu

245 250 255 Val Leu Arg Asp Ile Asp Leu Ala Leu Ala Tyr Ala Arg Ala Tyr Gly 260 265 270

Glu Cys Asp Arg Asp Val Trp Cys Asn Pro Ala His Ala Val Met Ser 275 280 285

Arg Lys Ile Gly Gin Leu Ile Gly Asp Leu Val Gin Gin Gly Ser Leu

290 295 300

Lys Glu Glu Val Leu Trp Asn Leu Ser Asp Arg Glu Phe Trp Glu Leu 305 310 315 320

Leu Lys Ser Lys Val Asp Ser Lys Gly Leu Glu Thr Ile Lys His Ile

325 330 335

Glu Ala Gly Pro His Ala Glu Asp Tyr His Arg Leu Pro Arg Gly Thr 340 345 350

Lys Ile Arg Thr Ile Asp Pro Asp Leu Leu Leu Pro Gly Ala Gly Gin 355 360 365

Pro Ser Pro Leu Ser Phe Val Lys Pro Glu Trp Ala Lys Glu Arg Gin

370 375 380

Asp Phe Ile Gin Ala Arg Gin Ala Leu Leu Ala Ser Phe Thr Val Tyr 385 390 395 400 Ile Asn Gly Gly Val Ser Trp Glu Gin Asp Ser Ile Gin Pro Gin Phe

405 410 415

Glu Asn Asn Asn Pro Ile Met Ser Glu Ala Leu Thr Asn Thr Asp Leu 420 425 430

Gin Gly Ala Leu Pro Leu Ile Ala Arg Gly Lys Val Arg Asp Leu Tyr

435 440 445

Asp Val Asp Glu Lys Thr Leu Leu Phe Val Ala Thr Asp Arg Ile Ser

450 455 460

Ala Tyr Asp Val Ile Met Glu Asn Gly Ile Pro Glu Lys Gly Ile Leu 465 470 475 480

Leu Thr Leu Cys Thr Lys Thr Trp Phe Lys Ile Leu Ser Asp Lys Ile

485 490 495

Pro Ser Leu Arg Thr His Phe Leu Thr Leu Asp Leu Pro Pro Gin Ile 500 505 510

Pro Glu Ser Leu Arg Pro Val Leu Gin Asn Arg Ser Met Gin Val Arg 515 520 525

Lys Leu Lys Ile Leu Pro Ile Glu Ala Ile Val Arg Gly Tyr Ile Thr

530 535 540

Gly Ser Ala Trp Asn Glu Tyr Lys Lys Ser Gly Thr Val His Gly Ile 545 550 555 560

Lys Val Ala Glu Gly Leu Arg Glu Ser Glu Ala Phe Pro Asp Gly Pro

565 570 575 Ile Tyr Thr Pro Ser Thr Lys Ala Glu Gin Gly Glu His Asp Glu Asn

580 585 590 Ile His Pro Asp Gin Ala Val Ala Ile Val Gly Glu Arg Tyr Ala Ser 595 600 605

Lys Ile Ala Ser Leu Ala Val Gin Leu Tyr Lys Val Ala His Glu Tyr

610 615 620

Ala Leu Thr Arg Gly Val Ile Ile Ala Asp Thr Lys Phe Glu Phe Gly 625 630 635 640

Leu Asp Pro Glu Thr Asp Glu Val Val Leu Ala Asp Glu Val Leu Thr

645 650 655 Pro Asp Ser Ser Arg Phe Trp Pro Lys Asp Ser Tyr Glu Ile Gly Arg 660 665 670

Gly Gin Gin Ser Phe Asp Lys Gin Phe Leu Arg Asp Trp Leu Thr Ser 675 680 685

Glu Gly Leu Lys Gly Lys Pro Gly Val Arg Met Thr Asp Glu Ile Ala

690 695 700

Gin Lys Thr Ser Ala Lys Tyr Arg Glu Ala Tyr Glu Arg Ile Thr Gly 705 710 715 720

Glu Thr Lys Val Pro Ala Val 725

<210> 189

<211> 750

<212> DNA

<213> Aspergillus oryzae

<220>

<221> promoter

<222> (1) • • (750)

<223> Aspergillus oryzae burden sensing promoter p (bibA)

<400> 189 ctatgtagaa tagatagtag taggggtgtc atcctccatt atctgacaag cagataccag 60 catgtggcca cgtgtagtga tgttgtgcaa gtcattaagc tgcagaggtc atgacttcac 120 agtgactaga gttgagtaac ttgagatatg gaaagtacac ggtgtactca aggtacagta 180 caattggagg ttgacgacgt cctttgtcca cgtatcctaa aggagcatat ggccgggggg 240 ctgaaaaata gggcaacttc aagaaagatt acctaacagg ggcttctagc caatgaaaca 300 catttctagt atgttcggtg gaccgcttgc agaagatcca tgaacacttt ggggcaattt 360 ctacggcaga atacagggcg gtggccaagg cggatgggtc attctggtcc ctgaaaggcg 420 atgcaccaat cacaacgcga ggagtaacct gcagagcgat ggccgtacca gccatgctgt 480 ccttttctct ggcctcaagc attttaaaaa agctgatctc ttcctctcac gtccttcgtt 540 cagcaacttc tctcttttgc ctcaactttc ccttccttcc cccttccatt ccgtcgcctg 600 tggttgggtt ctctttttct tccttttgct tctctttctt gaatacagga aactgtattg 660 aagcacaagg gatttactac caacacgtct cccgataatc acacgcgtgc cccttggcga 720 agaccaccac tcgatataca cataggcaca 750

<210> 190

<211> 750

<212> DNA <213> Aspergillus oryzae

<220>

<221> promoter

<222> (1) • • (750)

<223> Aspergillus oryzae burden sensing promoter p (clxA)

<400> 190 agtgtggtct gaagttttgc aaagtgagtg aggaggggaa aagaacgtcg gcggggagag 60 gggcaacgtg aagaactcca tttttttttt ttctggatat ggataattaa ctagtagcgg 120 gaagtagatc gcgcaatcgg tatttgcaag gatagtatgc aattacttcg aagattaaga 180 aaggaacaag aggaaaaata gaaaagaatt tatttatatt catttgaaat ttacattgac 240 tggattgaat tgtcttattt gaattgggtg actagcatgt ttgggagaag tgtactaata 300 tgaatgaatg gtatggagta ctacaaataa catacaggga cgctaagacc gtgacaacca 360 aagagagaaa acaaaaaaag caataaaacg gttctttaga atcaatggac actggacaga 420 acattggaga atctgccact aatcctgttt gggtcggtgg aactttaccc caaggggcaa 480 gcggcagcgg aagaggccag attgaactta agctaccttc ccagcttcat ataatcaacc 540 tccccaacac tcgtcttctc tttctcctct cctctctctt cttccttctg tcgtgcggtt 600 agcttgtggt acgctctcac cttgacactc ttttccccct cgaacctcta cggaggacca 660 attgcttgat cgtttgctta tttgtttctt tctttcctaa agctactacc gagtcgggct 720 tcttagtttg agagcccgtt atcattcatc 750

<210> 191

<211> 300

<212> DNA

<213> Corynebacterium glutamicum

<220>

<221> promoter

<222> (1) • • (300)

<223> C . glutamicum burden sensing promoter p (groES)

<400> 191 gcgaagacat cacgttggat gaaattgagc gaattaagga cggcattcgc gccgctcgta 60 actaccgcga tgattatcca gaggaattca acctgtggcg caacgctgta tataacctgc 120 gtacgcccta aagtttggct gccatgtgaa tttttagcac cctcaacagt tgagtgctgg 180 cactctcggg ggtagagtgc caaataggtt gtttgacaca cagttgttca cccgcgacga 240 cggctgtgct ggaaacccac aaccggcaca cacaaaattt ttctcatgga gggattcacc 300

<210> 192

<211> 300

<212> DNA

<213> Corynebacterium glutamicum <220>

<221> promoter

<222> (1) • • (300)

<223> C . glutamicum burden sensing promoter p (katA)

<400> 192 tttcccggat gaaatccgtg agagctgcgc gatcgccacg gccggcggct agggccagct 60 cggtgacgtg ggcgtcgtta cgctcttttg acttcacaat tgctcagaat accagttcag 120 accggggtca ccataaaggt gtgtaggggg tgtcaaagtg ggcaaaaagt atcatacttt 180 tgtctttttg ctggcagttt acgtaagggc tgattatttg gtggcgcgaa gcgacattct 240 gatgtagact atcaatcaga cttaatccat aggtaaccct cataaaagga aggaatgcta 300

<210> 193

<211> 300

<212> DNA

<213> Corynebacterium glutamicum

<220>

<221> promoter

<222> (1) • • (300)

<223> C . glutamicum burden sensing promoter p (cplX)

<400> 193 ggaggtggcg gtggtgcgac acacaacgcg cacgcccgat ggttggcgtt ccatcgcgct 60 aaactgggga tcacagtagc gttctactat ttctttgctg agtcgatgcg ggcgaaggcc 120 cctgtttgtg gtttcaattt cggttgcgcc gtgaataaat tcgggattca ttagcttaag 180 cgtaccgctc tgcagagttc acaggtaaag cgctaagatg gaacaaccca ttgccaatat 240 tgttggttag agttgtacgc agtaaatctt ttcaatcgtg gaagcgggtc tcacagtcta 300

<210> 194

<211> 300

<212> DNA

<213> Corynebacterium glutamicum

<220>

<221> promoter

<222> (1) • • (300)

<223> C . glutamicum burden sensing promoter p (mutM)

<400> 194 tggataccaa gttggcagag tttgaaacct cgctgtccac cacgttgcgt tctgtcactg 60 ctgatcgttc cgcactacgc cgaggagctg gagctactgg ccgcgagctg cgcgatgagc 120 aaccggcagc gcgtggtgaa tatgagcggg actacgagcg tgattatgaa cgaggctacg 180 aacgcgacga tcgagactac tagtttccaa gaaaccattt aggctttttc acctttccgt 240 ctagcgatcc tcatctgatg tatgtagtca cgatgaggta cgagtaggat cttcctggtc 300 <210> 195

<211> 4694

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4694 )

<223> plasmid pEG151

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> homology region; iscU upstream

<220>

<221> promoter

<222> (1833) . . (2128)

<223> burden addiction promoter p (groES)

<220>

<221> RBS

<222> (2129) . . (2134 )

<220>

<221> misc_feature

<222> (2134 ) . . (2134 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2144 ) . . (2621)

<223> homology region; iscU downstream

<400> 195 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttaatgcgga aaactgcatg atgatatatg 1860 tcaaaataat aaaccaatac tgttttctca aatggtatgt actcctttgt taagtgggtt 1920 tcgttcatct acagctattg taacataatc ggtacggggg tgaaaaagct aacggaaaag 1980 ggagcggaaa agaatgatgt aagcgtgaaa aattttttat cttatcactt gaaattggaa 2040 gggagattct ttattataag aattgtgtta gcactcttta gtgctgagtg ctaaaattac 2100 atattcatac tattgaggag gttatttcac gagntgaaat catatggctg cttcaacatt 2160 aacgatcaaa gatcttcacg ttgaaatcga agggaaagag atcttaaagg gtgtaaacct 2220 tgaaataaaa ggtggagaat tccacgcagt aatgggcccg aacggaactg gtaaatccac 2280 tttatcagct gctattatgg ggcatcctaa atatgaagta acaaaaggca gcatcacgct 2340 tgacggcaaa gatgtactgg agatggaagt ggacgagcgt gcgcaggcag gcttattcct 2400 tgcaatgcag tacccaagtg aaatcagcgg tgtgacaaat gccgacttcc ttcgctcagc 2460 aatcaacgcg cgcagagaag aaggcgatga aatttctctc atgaaattca tccgcaaaat 2520 ggacgaaaac atggagttcc ttgaaatgga ccctgaaatg gctcagcgct atcttaacga 2580 aggcttctca ggcggggaga aaaaacgcaa cgaaatcctt caattttatt gcaataacag 2640 gtgcttactt ttctggagtt ctttagcaaa tttttttatt agctgaactt agtattagtg 2700 gccatactcc tccaatccaa agctatttag aaagattact atatcctcaa acaggcggta 2760 accggcctct tcatcgggaa tgcgcgcgac cttcagcatc gccggcatgt ccccctggcg 2820 gacgggaagt atccagctcg aggtcgggcc gcgttgctgg cgtttttcca taggctccgc 2880 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2940 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 3000 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtgge gctttctcat 3060 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 3120 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 3180 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 3240 gcgaggtatg taggcggtge tacagagttc ttgaagtggt ggcctaacta cggctacact 3300 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3360 gatagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3420 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3480 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3540 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3600 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3660 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3720 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3780 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3840 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3900 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 3960 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 4020 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 4080 aagttggccg cagtgttatc actcatggtt atggcagcac tgeataatte tcttaetgte 4140 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 4200 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca 4260 catagcagaa ctttaaaagt gct cat cat t ggaaaacgtt cttcggggcg aaaactctca 4320 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 4380 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 4440 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4500 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4560 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 4620 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 4680 cgtcttcaag aatt 4694

<210> 196

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4697 )

<223> Plasmid pEG152

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> Homology region iscU upstream

<220>

<221> promoter

<222> (1833) . . (2131)

<223> burden addiction promoter p (ctsR)

<220>

<221> rbs

<222> (2132 ) . . (2137 )

<223> RBS

<220>

<221> misc_feature

<222> (2137 ) . . (2137 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> Homology region iscU downstream

<400> 196 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttgctcttca gcgccgatgg tagtcggggg 1860 tttccccctg tgagagtagg acgccgccaa gcaagcttaa acccagctca atgagctggg 1920 ttttttgtat tttggtttat tggtatcata aaattccact taactgtata atataataac 1980 tttataccga attttaaatc agcaatcagg ttttgtggac cgggaaaatg gaaataatga 2040 aggatagagc gagaaagttg aaaattctcg agaaacggct tatagtaaga ttaaagtcaa 2100 atatagtcaa agtcagtaaa ggagggggtt gacgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 197

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4697 )

<223> Plasmid pEG153

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> Homology region iscU upstream

<220>

<221> promoter

<222> (1833) . . (2131)

<223> burden addiction promoter p (dnaK)

<220>

<221> rbs

<222> (2132 ) . . (2137 )

<223> rbs

<220>

<221> misc_feature

<222> (2132 ) . . (2137 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> Homology region iscU downstream

<400> 197 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttggttcaat ccccgcacac gggaatttcg 1860 attaaaatcg gaaaagaaaa cgactatgaa gagatggaaa attgcagtct gattacggct 1920 tcttattccg tagaccagaa gcagatcggc tcaattgcga ttatcggccc gacccgcatg 1980 aattattcca gggttgtcag cctgcttcag catgtgactt cggacttgtc aaaagcatta 2040 acaagtctgt atgatgaata agggaatttt ggcaaatttt atcgaagggc agcacctgtc 2100 cttctcctta cactttgagg gaggtgaaca cacgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 198

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4697 )

<223> plasmid pEG154

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> homology region iscU upstream

<220>

<221> promoter

<222> (1833) . . (2131)

<223> burden addiction promoter p (perR)

<220>

<221> rbs

<222> (2132 ) . . (2137 )

<223> rbs

<220>

<221> misc_feature

<222> (2137 ) . . (2137 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> homology region iscU downstream

<400> 198 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttggaagcat ttgatgtgtg gaagctgaag 1860 aaaaacttcg gaaaagagta tatgggaatc gagcgttcca cttttttaat tgataaagaa 1920 ggccggctca ttaaagaatg gagaaaagta aaagtaaagg accatgtagc cgaagcgctt 1980 caaaccctca aagatatgtc tgaaaagtaa atctctatga gcctatgctt acttttaccc 2040 tgatatcaat tgacaaatta tcgtagaaag agttacacta attataaaca ttacaatgta 2100 agaatttttt ttagaagaga ggtgcatgac ggcgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 199

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4697 )

<223> plasmid pEG155

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> homology region iscU upstream

<220>

<221> promoter

<222> (1823) . . (2131)

<223> burden addiction promoter p (hrcA)

<220>

<221> promoter

<222> (1823) . . (2131)

<223> burden addiction promoter p (hrcA)

<220>

<221> rbs

<222> (2132 ) . . (2137 )

<223> rbs

<220>

<221> misc_feature

<222> (2132 ) . . (2137 ) <223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> homology region iscU downstream

<400> 199 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga tttgaggaag aaatgttttt aggtttgaga 1860 aaaacagccg gtgtcagcaa aaaacgattc gcagaaaaat acggccgttc cctggatgga 1920 cttttcccaa gcgtgttaaa agacctcgct gaaaaagggc tgattcacaa ttcggagtct 1980 gccgtttatt tgactcatca agggaaatta ttaggcaatg aagtttttgg cgcttttttg 2040 ggtgagttat aattgacatt tttcttgtgg tttgatactt ttgttataga attagcactc 2100 gcttattgag agtgctaaca gaggtgatga tgcgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 200

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4697 )

<223> plasmid pEG156

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> homology region iscU upstream

<220>

<221> promoter

<222> (1833) . . (2131)

<223> burden addiction promoter p (spxA)

<220> <221> rbs

<222> (2132 ) . . (2137 )

<223> rbs

<220>

<221> misc_feature

<222> (2137 ) . . (2137 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> homology region iscU downstream

<400> 200 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttgaaaacat tcatacttat aaagatgaga 1860 aatggtacgg agaatcgtac gaaaaaacgg atgaggtctt ggaaatcatc gatgaagaaa 1920 aacaaaaaaa tattttcgat cagttaagct aattgagaat aagaacatat catgaacatt 1980 aaaaatttgc tggattgaca catttttttc ttaggatact gttcacactt acttttttat 2040 agtataatac ctgtaaagat tcctttttta gagtaatttc aatttaagaa taaaatagat 2100 gttcatccta ctaattagag gagtgaagat gacgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 201

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4697 )

<223> plasmid pEG157

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> homology region iscU upstream <220>

<221> promoter

<222> (1833) . . (2131)

<223> burden addiction promoter p (sigB)

<220>

<221> rbs

<222> (2132 ) . . (2137 )

<223> rbs

<220>

<221> misc_feature

<222> (2132 ) . . (2137 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> homology region iscU downstream

<400> 201 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttaacaattc gatcagcatc tggaaaaggg 1860 cgatatgatt gttttatttt ctgacggcgt cacagaatgc agaacggaaa acggtttctt 1920 ggagcgtcct gatctgcaga agctcattga ggaacatatg tgttcctctg cgcaggaaat 1980 ggtcaaaaac atttatgaca gcctcctcaa attgcaggat tttcagcttc acgatgattt 2040 tacgttaatt gttttgcgga gaaaggttta acgtctgtca gacgagggta taaagcaact 2100 agtgatttga aggaaaattt gaggtgatac gacgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 202

<211> 4697

<212> DNA

<213> synthetic

<220>

<221> misc_feature <222> (1) . . (4697 )

<223> plasmid pEG158

<220>

<221> misc_feature

<222> (88) . . ( 651)

<223> homology region iscU upstream

<220>

<221> promoter

<222> (1833) . . (2131)

<223> burden adiction promoter p (yflT)

<220>

<221> rbs

<222> (2132 ) . . (2137 )

<223> rbs

<220>

<221> misc_feature

<222> (2137 ) . . (2137 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2147 ) . . (2624 )

<223> homology region iscU downstream

<400> 202 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttcat tgaggaattc ggaaacacca cgcattaaga 120 gaaggaatat tccgaaaaac cattctcatt tgttctgaaa ggatgctttc gtctcatctg 180 ccgctatttc acgccaaatc gtttttgcgg catccaacgc agcactggta caataaaaag 240 caaatatatg aagggattga ttcattattt gcagcagcca ggacagactg aacctcgatt 300 atacgtcaga aatggaaaaa gcgatgcagc tgtcacacgg aattggatac gaggaatatg 360 gacgccggct ggaaacaaga atgaaagtag agcggcagag agagcttgat tatgaaaaaa 420 gcaagcggat ttctgcggga gcttattgag aaaatcaatc ttttaaaaaa gggtgggaat 480 ccactctttt tctttttcaa tcgctttaat aaagtgctga tctttgttaa acttgaggaa 540 tgtgcaaggc acgataaaaa gttgtattta cttttcattt gttataaaat tagaatgaga 600 ataaaatgag aataatgatt atatattttt ggaggtatag atatggctgc tagttcggat 660 tagatccccc tatgcaaggg tttattgttt tctaaaatct gattaccaat tagaatgaat 720 atttcccaaa tattaaataa taaaacaaaa aaattgaaaa aagtgtttcc accatttttt 780 caattttttt ataatttttt taatctgtta tttaaatagt ttatagttaa atttacattt 840 tcattagtcc attcaatatt ctctccaaga taactacgaa ctgctaacaa aattctctcc 900 ctatgttcta atggagaaga ttcagccact gcatttcccg caatatcttt tggtatgatt 960 ttacccgtgt ccatagttaa aatcatacgg cataaagtta atatagagtt ggtttcatca 1020 tcctgataat tatctattaa ttcctctgac gaatccataa tggctcttct cacatcagaa 1080 aatggaatat caggtagtaa ttcctctaag tcataatttc cgtatattct tttatttttt 1140 cgttttgctt ggtaaagcat tatggttaaa tctgaattta attccttctg aggaatgtat 1200 ccttgttcat aaagctcttg taaccattct ccataaataa attcttgttt gggaggatga 1260 ttccacggta ccatttcttg ctgaataata attgttaatt caatatatcg taagttgctt 1320 ttatctccta ttttttttga aataggtcta attttttgta taagtatttc tttactttga 1380 tctgtcaatg gttcagatac gacgactaaa aagtcaagat cactatttgg ttttagtcca 1440 ctctcaactc ctgatccaaa catgtaagta ccaataaggt tattttttaa atgtttccga 1500 agtatttttt tcactttatt aatttgttcg tatgtattca aatatatcct cctcactatt 1560 ttgattagta cctattttat atccatagtt gttaattaaa taaacttaat ttagtttatt 1620 tatagatttc attggcttct aaatttttta tctagataat aattatttta gttaatttta 1680 ttctagatta tatatgatat gatctttcat ttccataaaa ctaaagtaag tgtaaaccta 1740 ttcattgttt taaaaatatc tcttgccagt cacgttacgt tattagttat agttattata 1800 acatgtattc acgaacgaaa atcgcatcga ttgactaaat cactcacaat ccaagcattg 1860 tattgctgtt ctggtgaaaa gtattgcatt agacatacct cctgctcgta cggataaagg 1920 cagcgtttca tggtcgtgtg ctccgtgcag cggcttctcc ttaattttga tttttctgaa 1980 aataggtccc gttcctatca ctttaccatg gacggaaaac aaatagctac taccattcct 2040 cctgtttttc tcttcaatgt tctggaatct gtttcaggta cagacgatcg ggtatgaaag 2100 aaatatagaa aacatgaagg aggaatatcg accgagntga aatcatatgg ctgcttcaac 2160 attaacgatc aaagatcttc acgttgaaat cgaagggaaa gagatcttaa agggtgtaaa 2220 ccttgaaata aaaggtggag aattccacgc agtaatgggc ccgaacggaa ctggtaaatc 2280 cactttatca gctgctatta tggggcatcc taaatatgaa gtaacaaaag gcagcatcac 2340 gcttgacggc aaagatgtac tggagatgga agtggacgag cgtgcgcagg caggcttatt 2400 ccttgcaatg cagtacccaa gtgaaatcag cggtgtgaca aatgccgact tccttcgctc 2460 agcaatcaac gcgcgcagag aagaaggcga tgaaatttct ctcatgaaat tcatccgcaa 2520 aatggacgaa aacatggagt tccttgaaat ggaccctgaa atggctcagc gctatcttaa 2580 cgaaggcttc tcaggcgggg agaaaaaacg caacgaaatc cttcaatttt attgcaataa 2640 caggtgctta cttttctgga gttctttagc aaattttttt attagctgaa cttagtatta 2700 gtggccatac tcctccaatc caaagctatt tagaaagatt actatatcct caaacaggcg 2760 gtaaccggcc tcttcatcgg gaatgcgcgc gaccttcagc atcgccggca tgtccccctg 2820 gcggacggga agtatccagc tcgaggtcgg gccgcgttgc tggcgttttt ccataggctc 2880 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2940 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 3000 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 3060 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 3120 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3180 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3240 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3300 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3360 gttgatagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3420 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3480 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3540 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3600 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3660 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3720 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3780 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3840 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3900 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca 3960 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 4020 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 4080 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 4140 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4200 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg 4260 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4320 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4380 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4440 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4500 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4560 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4620 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4680 tttcgtcttc aagaatt 4697

<210> 203 <211> 4696

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4696)

<223> plasmid pEG159

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region aaC upstream

<220>

<221> promoter

<222> (1858) . . (2153)

<223> burden adiction promoter p (groES)

<220>

<221> rbs

<222> (2154 ) . . (2159)

<223> rbs

<220>

<221> misc_feature

<222> (2154 ) . . (2159)

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2169) . . (2623)

<223> homology region aaC downstream

<400> 203 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattaat 1860 gcggaaaact gcatgatgat atatgtcaaa ataataaacc aatactgttt tctcaaatgg 1920 tatgtactcc tttgttaagt gggtttcgtt catctacagc tattgtaaca taatcggtac 1980 gggggtgaaa aagctaacgg aaaagggagc ggaaaagaat gatgtaagcg tgaaaaattt 2040 tttatcttat cacttgaaat tggaagggag attctttatt ataagaattg tgttagcact 2100 ctttagtgct gagtgctaaa attacatatt catactattg aggaggttat ttcacgagnt 2160 gaaatcatat gttaaatatc aaagaaatcc acgagctgat taaagcaatt gacgagtcta 2220 caattgacga attcgtatat gaaaatgaag gtgtatcctt aaaactgaaa aaacacgaag 2280 caggcacggt tcaagtcatg cagcaggcac cggcagcacc tgtacaagca caggctccgc 2340 aggcagttca gccgcaagcg cagcaagcag cggcacctgc ccaagaagca ccaaagcaag 2400 atgagaatct gcataaaatc acttcaccaa tggtaggaac attttatgct tcttcatcac 2460 cggaagctgg cccgtatgta acagccggtt caaaagtaaa tgaaaacaca gttgtctgca 2520 ttgtagaagc gatgaagctt ttcaacgaaa tcgaagcaga agtgaaaggc gaaatcgttg 2580 aagtattagt agaaaacggc cagctggtcg aatacggaca accaatttta ttgcaataac 2640 aggtgcttac ttttctggag ttctttagca aattttttta ttagctgaac ttagtattag 2700 tggccatact cctccaatcc aaagctattt agaaagatta ctatatcctc aaacaggcgg 2760 taaccggcct cttcatcggg aatgcgcgcg accttcagca tcgccggcat gtccccctgg 2820 cggacgggaa gtatccagct cgaggtcggg ccgcgttgct ggcgtttttc cataggctcc 2880 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 2940 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3000 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3060 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3120 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3180 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3240 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3300 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3360 ttgatagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3420 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3480 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 3540 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 3600 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 3660 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 3720 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 3780 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 3840 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 3900 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac 3960 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4020 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4080 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4140 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4200 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc 4260 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4320 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4380 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4440 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 4500 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 4560 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 4620 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 4680 ttcgtcttca agaatt 4696

<210> 204

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG160

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream

<220>

<221> promoter

<222> (1858) . . (2156)

<223> burden adiction promoter p (ctsR)

<220>

<221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream

<400> 204 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattgct 1860 cttcagcgcc gatggtagtc gggggtttcc ccctgtgaga gtaggacgcc gccaagcaag 1920 cttaaaccca gctcaatgag ctgggttttt tgtattttgg tttattggta tcataaaatt 1980 ccacttaact gtataatata ataactttat accgaatttt aaatcagcaa tcaggttttg 2040 tggaccggga aaatggaaat aatgaaggat agagcgagaa agttgaaaat tctcgagaaa 2100 cggcttatag taagattaaa gtcaaatata gtcaaagtca gtaaaggagg gggttgacga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 205

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG161

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream

<220>

<221> promoter

<222> (1858) . . (2156)

<223> burden adiction promoter p (dnaK)

<220>

<221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream

<400> 205 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattggt 1860 tcaatccccg cacacgggaa tttcgattaa aatcggaaaa gaaaacgact atgaagagat 1920 ggaaaattgc agtctgatta cggcttctta ttccgtagac cagaagcaga tcggctcaat 1980 tgcgattatc ggcccgaccc gcatgaatta ttccagggtt gtcagcctgc ttcagcatgt 2040 gacttcggac ttgtcaaaag cattaacaag tctgtatgat gaataaggga attttggcaa 2100 attttatcga agggcagcac ctgtccttct ccttacactt tgagggaggt gaacacacga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 206

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG162

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream

<220>

<221> promoter

<222> (1858) . . (2156)

<223> burden adictionpromoter p (perR)

<220>

<221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream

<400> 206 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattgga 1860 agcatttgat gtgtggaagc tgaagaaaaa cttcggaaaa gagtatatgg gaatcgagcg 1920 ttccactttt ttaattgata aagaaggccg gctcattaaa gaatggagaa aagtaaaagt 1980 aaaggaccat gtagccgaag cgcttcaaac cctcaaagat atgtctgaaa agtaaatctc 2040 tatgagccta tgcttacttt taccctgata tcaattgaca aattatcgta gaaagagtta 2100 cactaattat aaacattaca atgtaagaat tttttttaga agagaggtgc atgacggcga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 207

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG163

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream

<220>

<221> promoter

<222> (1858) . . (2156)

<223> burden adiction promoter p (hrcA)

<220>

<221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 )

<223> N is A, T , C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream <400> 207 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgatttga 1860 ggaagaaatg tttttaggtt tgagaaaaac agccggtgtc agcaaaaaac gattcgcaga 1920 aaaatacggc cgttccctgg atggactttt cccaagcgtg ttaaaagacc tcgctgaaaa 1980 agggctgatt cacaattcgg agtctgccgt ttatttgact catcaaggga aattattagg 2040 caatgaagtt tttggcgctt ttttgggtga gttataattg acatttttct tgtggtttga 2100 tacttttgtt atagaattag cactcgctta ttgagagtgc taacagaggt gatgatgcga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 208

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG164

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream

<220>

<221> promoter

<222> (1858) . . (2156)

<223> burden ediction promoter p (spxA)

<220>

<221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 ) <223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream

<400> 208 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattgaa 1860 aacattcata cttataaaga tgagaaatgg tacggagaat cgtacgaaaa aacggatgag 1920 gtcttggaaa tcatcgatga agaaaaacaa aaaaatattt tcgatcagtt aagctaattg 1980 agaataagaa catatcatga acattaaaaa tttgctggat tgacacattt ttttcttagg 2040 atactgttca cacttacttt tttatagtat aatacctgta aagattcctt ttttagagta 2100 atttcaattt aagaataaaa tagatgttca tcctactaat tagaggagtg aagatgacga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 209

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG165

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream

<220>

<221> promoter

<222> (1858) . . (2156)

<223> burden adiction promoter p (sigB)

<220> <221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream

<400> 209 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattaac 1860 aattcgatca gcatctggaa aagggcgata tgattgtttt attttctgac ggcgtcacag 1920 aatgcagaac ggaaaacggt ttcttggagc gtcctgatct gcagaagctc attgaggaac 1980 atatgtgttc ctctgcgcag gaaatggtca aaaacattta tgacagcctc ctcaaattgc 2040 aggattttca gcttcacgat gattttacgt taattgtttt gcggagaaag gtttaacgtc 2100 tgtcagacga gggtataaag caactagtga tttgaaggaa aatttgaggt gatacgacga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 210

<211> 4699

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (4699)

<223> plasmid pEG166

<220>

<221> misc_feature

<222> (88) . . ( 676)

<223> homology region accC upstream <220>

<221> promoter

<222> (1858) . . (2156)

<223> burden adiction promoter p (yflT)

<220>

<221> rbs

<222> (2157 ) . . (2162 )

<223> rbs

<220>

<221> misc_feature

<222> (2162 ) . . (2162 )

<223> N is A, T, C or G

<220>

<221> misc_feature

<222> (2172 ) . . (2626)

<223> homology region accC downstream

<400> 210 aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60 gggatttttg actccgaagt aagtcttagg gaacgaaaga ttcatcagca gacaaggaaa 120 catccgctga agccagtgaa aaaggaactg ttgttacgga aacagccgat gatgatttgt 180 ttacaacgta ccgtcttgat ttagaagatg ccagaagcaa agaaagagaa gagctgaatg 240 ccattgtgtc aagcgatgat gcaacagcca aggagaaaag cgaagcatac gataaaatga 300 cggctctcag tgaagtggaa ggaacagaaa aacagctgga aacgctgatt aaaacacaag 360 gttacgagga tgcgcttgtt aatgctgaag gagataaaat caatattaca gtcaaatcag 420 acaaacactc taaatcgaag gcgacagcca ttatagacct tgtggcaaaa gaaatcaaaa 480 caatgaaaga tgtcgctgtc acatttgaac cctctaaata agaatgaggg aaaaaagccc 540 gctaaacaag cgggcttttt gcgttgctgt catattagag ttgaattcaa aagtccgctc 600 ctgtaagatg aacatagtat gtacttttag tagtaaccta taaaaaaaga aatttcatac 660 ataggagtgc gattgaagtt cggattagat ccccctatgc aagggtttat tgttttctaa 720 aatctgatta ccaattagaa tgaatatttc ccaaatatta aataataaaa caaaaaaatt 780 gaaaaaagtg tttccaccat tttttcaatt tttttataat ttttttaatc tgttatttaa 840 atagtttata gttaaattta cattttcatt agtccattca atattctctc caagataact 900 acgaactgct aacaaaattc tctccctatg ttctaatgga gaagattcag ccactgcatt 960 tcccgcaata tcttttggta tgattttacc cgtgtccata gttaaaatca tacggcataa 1020 agttaatata gagttggttt catcatcctg ataattatct attaattcct ctgacgaatc 1080 cataatggct cttctcacat cagaaaatgg aatatcaggt agtaattcct ctaagtcata 1140 atttccgtat attcttttat tttttcgttt tgcttggtaa agcattatgg ttaaatctga 1200 atttaattcc ttctgaggaa tgtatccttg ttcataaagc tcttgtaacc attctccata 1260 aataaattct tgtttgggag gatgattcca cggtaccatt tcttgctgaa taataattgt 1320 taattcaata tatcgtaagt tgcttttatc tcctattttt tttgaaatag gtctaatttt 1380 ttgtataagt atttctttac tttgatctgt caatggttca gatacgacga ctaaaaagtc 1440 aagatcacta tttggtttta gtccactctc aactcctgat ccaaacatgt aagtaccaat 1500 aaggttattt tttaaatgtt tccgaagtat ttttttcact ttattaattt gttcgtatgt 1560 attcaaatat atcctcctca ctattttgat tagtacctat tttatatcca tagttgttaa 1620 ttaaataaac ttaatttagt ttatttatag atttcattgg cttctaaatt ttttatctag 1680 ataataatta ttttagttaa ttttattcta gattatatat gatatgatct ttcatttcca 1740 taaaactaaa gtaagtgtaa acctattcat tgttttaaaa atatctcttg ccagtcacgt 1800 tacgttatta gttatagtta ttataacatg tattcacgaa cgaaaatcgc atcgattgac 1860 taaatcactc acaatccaag cattgtattg ctgttctggt gaaaagtatt gcattagaca 1920 tacctcctgc tcgtacggat aaaggcagcg tttcatggtc gtgtgctccg tgcagcggct 1980 tctccttaat tttgattttt ctgaaaatag gtcccgttcc tatcacttta ccatggacgg 2040 aaaacaaata gctactacca ttcctcctgt ttttctcttc aatgttctgg aatctgtttc 2100 aggtacagac gatcgggtat gaaagaaata tagaaaacat gaaggaggaa tatcgaccga 2160 gntgaaatca tatgttaaat atcaaagaaa tccacgagct gattaaagca attgacgagt 2220 ctacaattga cgaattcgta tatgaaaatg aaggtgtatc cttaaaactg aaaaaacacg 2280 aagcaggcac ggttcaagtc atgcagcagg caccggcagc acctgtacaa gcacaggctc 2340 cgcaggcagt tcagccgcaa gcgcagcaag cagcggcacc tgcccaagaa gcaccaaagc 2400 aagatgagaa tctgcataaa atcacttcac caatggtagg aacattttat gcttcttcat 2460 caccggaagc tggcccgtat gtaacagccg gttcaaaagt aaatgaaaac acagttgtct 2520 gcattgtaga agcgatgaag cttttcaacg aaatcgaagc agaagtgaaa ggcgaaatcg 2580 ttgaagtatt agtagaaaac ggccagctgg tcgaatacgg acaaccaatt ttattgcaat 2640 aacaggtgct tacttttctg gagttcttta gcaaattttt ttattagctg aacttagtat 2700 tagtggccat actcctccaa tccaaagcta tttagaaaga ttactatatc ctcaaacagg 2760 cggtaaccgg cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc 2820 tggcggacgg gaagtatcca gctcgaggtc gggccgcgtt gctggcgttt ttccataggc 2880 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3360 gagttgatag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3420 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 3720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 3780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 3840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 3900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt 3960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4140 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4200 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 4260 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4320 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4380 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4440 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4500 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4560 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4620 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 4680 cctttcgtct tcaagaatt 4699

<210> 211

<211> 783

<212> DNA

<213> synthetic

<220>

<221> misc_feature <222> (1) • • (783)

<223> Gene encoding SpcR (resistance marker)

<400> 211 gtactaatca aaatagtgag gaggatatat ttgaatacat acgaacaaat taataaagtg 60 aaaaaaatac ttcggaaaca tttaaaaaat aaccttattg gtacttacat gtttggatca 120 ggagttgaga gtggactaaa accaaatagt gatcttgact ttttagtcgt cgtatctgaa 180 ccattgacag atcaaagtaa agaaatactt atacaaaaaa ttagacctat ttcaaaaaaa 240 ataggagata aaagcaactt acgatatatt gaattaacaa ttattattca gcaagaaatg 300 gtaccgtgga atcatcctcc caaacaagaa tttatttatg gagaatggtt acaagagctt 360 tatgaacaag gatacattcc tcagaaggaa ttaaattcag atttaaccat aatgctttac 420 caagcaaaac gaaaaaataa aagaatatac ggaaattatg acttagagga attactacct 480 gatattccat tttctgatgt gagaagagcc attatggatt cgtcagagga attaatagat 540 aattatcagg atgatgaaac caactctata ttaactttat gccgtatgat tttaactatg 600 gacacgggta aaatcatacc aaaagatatt gcgggaaatg cagtggctga atcttctcca 660 ttagaacata gggagagaat tttgttagca gttcgtagtt atcttggaga gaatattgaa 720 tggactaatg aaaatgtaaa tttaactata aactatttaa ataacagatt aaaaaaatta 780 taa 783

<210> 212

<211> 296

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) • • (296)

<223> Bacillus bruden sensing promoter p (groES)

<400> 212 aatgcggaaa actgcatgat gatatatgtc aaaataataa accaatactg ttttctcaaa 60 tggtatgtac tcctttgtta agtgggtttc gttcatctac agctattgta acataatcgg 120 tacgggggtg aaaaagctaa cggaaaaggg agcggaaaag aatgatgtaa gcgtgaaaaa 180 ttttttatct tatcacttga aattggaagg gagattcttt attataagaa ttgtgttagc 240 actctttagt gctgagtgct aaaattacat attcatacta ttgaggaggt tatttc 296

<210> 213

<211> 299

<212> DNA

<213> Bacillus

<220> <221> promoter

<222> (1) • • (299)

<223> Bacillus burden sensing promoter p (ctsR)

<400> 213 gctcttcagc gccgatggta gtcgggggtt tccccctgtg agagtaggac gccgccaagc 60 aagcttaaac ccagctcaat gagctgggtt ttttgtattt tggtttattg gtatcataaa 120 attccactta actgtataat ataataactt tataccgaat tttaaatcag caatcaggtt 180 ttgtggaccg ggaaaatgga aataatgaag gatagagcga gaaagttgaa aattctcgag 240 aaacggctta tagtaagatt aaagtcaaat atagtcaaag tcagtaaagg agggggttg 299

<210> 214

<211> 299

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) • • (299)

<223> Bacillus burden sensing promoter p (dnaK)

<400> 214 ggttcaatcc ccgcacacgg gaatttcgat taaaatcgga aaagaaaacg actatgaaga 60 gatggaaaat tgcagtctga ttacggcttc ttattccgta gaccagaagc agatcggctc 120 aattgcgatt atcggcccga cccgcatgaa ttattccagg gttgtcagcc tgcttcagca 180 tgtgacttcg gacttgtcaa aagcattaac aagtctgtat gatgaataag ggaattttgg 240 caaattttat cgaagggcag cacctgtcct tctccttaca ctttgaggga ggtgaacac 299

<210> 215

<211> 299

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) • • (299)

<223> Bacillus burden addiction sensing p (perR)

<400> 215 ggaagcattt gatgtgtgga agctgaagaa aaacttcgga aaagagtata tgggaatcga 60 gcgttccact tttttaattg ataaagaagg ccggctcatt aaagaatgga gaaaagtaaa 120 agtaaaggac catgtagccg aagcgcttca aaccctcaaa gatatgtctg aaaagtaaat 180 ctctatgagc ctatgcttac ttttaccctg atatcaattg acaaattatc gtagaaagag 240 ttacactaat tataaacatt acaatgtaag aatttttttt agaagagagg tgcatgacg 299

<210> 216 <211> 299

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) • • (299)

<223> Bacillus burden sensing promoter p (hrcA)

<400> 216 tgaggaagaa atgtttttag gtttgagaaa aacagccggt gtcagcaaaa aacgattcgc 60 agaaaaatac ggccgttccc tggatggact tttcccaagc gtgttaaaag acctcgctga 120 aaaagggctg attcacaatt cggagtctgc cgtttatttg actcatcaag ggaaattatt 180 aggcaatgaa gtttttggcg cttttttggg tgagttataa ttgacatttt tcttgtggtt 240 tgatactttt gttatagaat tagcactcgc ttattgagag tgctaacaga ggtgatgat 299

<210> 217

<211> 299

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) • • (299)

<223> Bacillus burden sensing promoter p (spxA)

<400> 217 gaaaacattc atacttataa agatgagaaa tggtacggag aatcgtacga aaaaacggat 60 gaggtcttgg aaatcatcga tgaagaaaaa caaaaaaata ttttcgatca gttaagctaa 120 ttgagaataa gaacatatca tgaacattaa aaatttgctg gattgacaca tttttttctt 180 aggatactgt tcacacttac ttttttatag tataatacct gtaaagattc cttttttaga 240 gtaatttcaa tttaagaata aaatagatgt tcatcctact aattagagga gtgaagatg 299

<210> 218

<211> 299

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) • • (299)

<223> Bacillus burden sensing promoter p (sigB)

<400> 218 aacaattcga tcagcatctg gaaaagggcg atatgattgt tttattttct gacggcgtca 60 cagaatgcag aacggaaaac ggtttcttgg agcgtcctga tctgcagaag ctcattgagg 120 aacatatgtg ttcctctgcg caggaaatgg tcaaaaacat ttatgacagc ctcctcaaat 180 tgcaggattt tcagcttcac gatgatttta cgttaattgt tttgcggaga aaggtttaac 240 gtctgtcaga cgagggtata aagcaactag tgatttgaag gaaaatttga ggtgatacg 299

<210> 219

<211> 299

<212> DNA

<213> Bacillus

<220>

<221> promoter

<222> (1) . . (299)

<223> Bacillus burden sensing promoter p (yflT)

<400> 219 gactaaatca ctcacaatcc aagcattgta ttgctgttct ggtgaaaagt attgcattag 60 acatacctcc tgctcgtacg gataaaggca gcgtttcatg gtcgtgtgct ccgtgcagcg 120 gcttctcctt aattttgatt tttctgaaaa taggtcccgt tcctatcact ttaccatgga 180 cggaaaacaa atagctacta ccattcctcc tgtttttctc ttcaatgttc tggaatctgt 240 ttcaggtaca gacgatcggg tatgaaagaa atatagaaaa catgaaggag gaatatcga 299

<210> 220

<211> 651

<212> DNA

<213> synthetic

<220>

<221> CDS

<222> (1) . . ( 651)

<223> chlR gene encoding camR resistance marker

<400> 220 atg aac ttt aat aaa att gat tta gac aat tgg aag aga aaa gag ata 48

Met Asn Phe Asn Lys Ile Asp Leu Asp Asn Trp Lys Arg Lys Glu Ile 1 5 10 15 ttt aat cat tat ttg aac caa caa acg act ttt agt ata acc aca gaa 96

Phe Asn His Tyr Leu Asn Gin Gin Thr Thr Phe Ser Ile Thr Thr Glu

20 25 30 att gat att agt gtt tta tac cga aac ata aaa caa gaa gga tat aaa 144 Ile Asp Ile Ser Val Leu Tyr Arg Asn Ile Lys Gin Glu Gly Tyr Lys

35 40 45 ttt tac cct gca ttt att ttc tta gtg aca agg gtg ata aac tca aat 192

Phe Tyr Pro Ala Phe Ile Phe Leu Val Thr Arg Val Ile Asn Ser Asn

50 55 60 aca gct ttt aga act ggt tac aat agc gac gga gag tta ggt tat tgg 240

Thr Ala Phe Arg Thr Gly Tyr Asn Ser Asp Gly Glu Leu Gly Tyr Trp

65 70 75 80 gat aag tta gag cca ctt tat aca att ttt gat ggt gta tct aaa aca 288

Asp Lys Leu Glu Pro Leu Tyr Thr Ile Phe Asp Gly Val Ser Lys Thr

85 90 95 ttc tct ggt att tgg act cct gta aag aat gac ttc aaa gag ttt tat 336 Phe Ser Gly Ile Trp Thr Pro Val Lys Asn Asp Phe Lys Glu Phe Tyr 100 105 110 gat tta tac ctt tct gat gta gag aaa tat aat ggt tcg ggg aaa ttg 384

Asp Leu Tyr Leu Ser Asp Val Glu Lys Tyr Asn Gly Ser Gly Lys Leu

115 120 125 ttt ccc aaa aca cct ata cct gaa aat gct ttt tct ctt tct att att 432

Phe Pro Lys Thr Pro Ile Pro Glu Asn Ala Phe Ser Leu Ser Ile Ile

130 135 140 cca tgg act tca ttt act ggg ttt aac tta aat atc aat aat aat agt 480

Pro Trp Thr Ser Phe Thr Gly Phe Asn Leu Asn Ile Asn Asn Asn Ser

145 150 155 160 aat tac ctt cta ccc att att aca gca gga aaa ttc att aat aaa ggt 528

Asn Tyr Leu Leu Pro Ile Ile Thr Ala Gly Lys Phe Ile Asn Lys Gly

165 170 175 aat tca ata tat tta ccg cta tct tta cag gta cat cat tct gtt tgt 576

Asn Ser Ile Tyr Leu Pro Leu Ser Leu Gin Val His His Ser Val Cys

180 185 190 gat ggt tat cat gca gga ttg ttt atg aac tct att cag gaa ttg tca 624

Asp Gly Tyr His Ala Gly Leu Phe Met Asn Ser Ile Gin Glu Leu Ser

195 200 205 gat agg cct aat gac tgg ctt tta taa 651

Asp Arg Pro Asn Asp Trp Leu Leu

210 215

<210> 221

<211> 216

<212> PRT

<213> synthetic

<400> 221

Met Asn Phe Asn Lys Ile Asp Leu Asp Asn Trp Lys Arg Lys Glu Ile

1 5 10 15

Phe Asn His Tyr Leu Asn Gin Gin Thr Thr Phe Ser Ile Thr Thr Glu 20 25 30 Ile Asp Ile Ser Val Leu Tyr Arg Asn Ile Lys Gin Glu Gly Tyr Lys 35 40 45

Phe Tyr Pro Ala Phe Ile Phe Leu Val Thr Arg Val Ile Asn Ser Asn 50 55 60

Thr Ala Phe Arg Thr Gly Tyr Asn Ser Asp Gly Glu Leu Gly Tyr Trp 65 70 75 80

Asp Lys Leu Glu Pro Leu Tyr Thr Ile Phe Asp Gly Val Ser Lys Thr 85 90 95 Phe Ser Gly Ile Trp Thr Pro Val Lys Asn Asp Phe Lys Glu Phe Tyr 100 105 110

Asp Leu Tyr Leu Ser Asp Val Glu Lys Tyr Asn Gly Ser Gly Lys Leu 115 120 125

Phe Pro Lys Thr Pro Ile Pro Glu Asn Ala Phe Ser Leu Ser Ile Ile

130 135 140

Pro Trp Thr Ser Phe Thr Gly Phe Asn Leu Asn Ile Asn Asn Asn Ser 145 150 155 160

Asn Tyr Leu Leu Pro Ile Ile Thr Ala Gly Lys Phe Ile Asn Lys Gly

165 170 175

Asn Ser Ile Tyr Leu Pro Leu Ser Leu Gin Val His His Ser Val Cys 180 185 190

Asp Gly Tyr His Ala Gly Leu Phe Met Asn Ser Ile Gin Glu Leu Ser 195 200 205

Asp Arg Pro Asn Asp Trp Leu Leu

210 215

<210> 222

<211> 10063

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (10063)

<223> plasmid pEG062

<220>

<221> misc_feature

<222> (1) . . ( 637 )

<223> homology region amyE upstream

<220>

<221> misc_feature

<222> (789) . . (1583)

<223> resistance marker KanR (on complementary strand)

<220>

<221> terminator

<222> (1744 ) . . (1796)

<223> terminator T7

<220>

<221> promoter

<222> (1866) . . (2655) <223> promoter P3P

<220>

<221> misc_feature

<222> (2680) . . (3861)

<223> gene encoding IgA-V3

<220>

<221> misc_feature

<222> (3862 ) . . (3867 )

<223> glycine linker

<220>

<221> misc_feature

<222> (3868) . . (4584 )

<223> gene encoding sfGFP

<220>

<221> misc_feature

<222> (4585) . . (4602 )

<223> purification tag 6-his

<220>

<221> terminator

<222> (4614 ) . . (4682 )

<223> terminator

<220>

<221> misc_feature

<222> (5070) . . ( 6262 )

<223> homology region amyE downstream

<220>

<221> misc_feature

<222> (7207 ) . . (7989)

<223> spc gene encoding resistance marker spectinomycun adenyltransferase

<220>

<221> misc_feature

<222> (8995) . . ( 9855)

<223> bla gene encoding bcta-lactamase for ampicillin resistance (on complementary strand)

<400> 222 ttaacaaaat tctccagtct tcacatcggt ttgaaaggag gaagcggaag aatgaagtaa 60 gagggatttt tgactccgaa gtaagtcttc aaaaaatcaa ataaggagtg tcaagaatgt 120 ttgcaaaacg attcaaaacc tctttactgc cgttattcgc tggattttta ttgctgtttc 180 atttggttct ggcaggaccg gcggctgcga gtgctgaaac ggcgaacaaa tcgaatgagc 240 ttacagcacc gtcgatcaaa agcggaacca ttcttcatgc atggaattgg tcgttcaata 300 cgttaaaaca caatatgaag gatattcatg atgcaggata tacagccatt cagacatctc 360 cgattaacca agtaaaggaa gggaatcaag gagataaaag catgtcgaac tggtactggc 420 tgtatcagcc gacatcgtat caaattggca accgttactt aggtactgaa caagaattta 480 aagaaatgtg tgcagccgct gaagaatatg gcataaaggt cattgttgac gcggtcatca 540 atcataccac cagtgattat gccgcgattt ccaatgaggt taagagtatt ccaaactgga 600 cacatggaaa cacacaaatt aaaaactggt ctgatcggat cctagaagct tatcgaattc 660 tcatgtttga cagcttatca tcggcaatag ttcggataat gagtttataa aataaaaaaa 720 gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg aagcttttta gacatctaaa 780 tctaggtact aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc 840 ttgatcccca gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg 900 atcgaccgga cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga 960 tcaataaagc cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg 1020 tgggaaaaga caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc 1080 ggatctttaa atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta 1140 ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc 1200 gatatgtcga tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca 1260 gggctttgtt catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc 1320 agattgctcc agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct 1380 tccagccata gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg 1440 ctgtccgtca tttttaaata taggttttca ttttctccca ccagcttata taccttagca 1500 ggagacattc cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc 1560 ggtgatattc tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga 1620 taccccaaga agctaattat aacaagacga actccaattc actgttcctt gcattctaaa 1680 accttaaata ccagaaaaca gacccaggtc tttttcaaag ttgttttgaa agttggcccg 1740 catcccccct agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttggagc 1800 tcaaacaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgcg 1860 tctattcttt tggaagaaaa tatagggaaa atggtatttg ttaaaaattc tgaatattta 1920 tacaatatca tatgtttcac attgaaatat gctgtccaga ctgtccgctg tgtaaaaaat 1980 aggaataaag gggggttgtt attattttac tgatatgtaa aatataattt gtataagaaa 2040 attcgaaacg taagatgaaa ccttagataa aagtgctttt tttgttgcaa ttgaagaatt 2100 attaatgtta agcttaatta aagataatat ctttgaattg taacgcccct caaaagtaag 2160 aactacaaaa aaagaatacg ttatatagaa atatgtttga accttcttca gattacaaat 2220 atattcggac ggactctacc tcaaatgctt atctaactat agaatgacat acaagcacaa 2280 ccttgaaaat ttgaaaatat aactaccaat gaacttgttc atgtgaatta tcgctgtatt 2340 taattttctc aattcaatat ataatatgcc aatacattgt tacaagtaga aattaagaca 2400 cccttgatag ccttactata cctaacatga tgtagtatta aatgaatatg taaatatatt 2460 tatgataaga agcgacttat ttataatcat tacatatttt tctattggaa tgattaagat 2520 tccaatagaa tagtgtataa attatttatc ttgaaaggag ggatgcctaa aaacgaagaa 2580 cattaaaaac atatatttgc accgtctaat ggatttatga aaaatcattt tatcagtttg 2640 aaaattatgt attatgatta actaataagg aggacaaaca tgatccaaaa acgcaaacgc 2700 acagttagct ttagactggt tctgatgtgc acactgctgt ttgtttcact gccgattaca 2760 aaaacaagcg cacaagttca actgcaagaa tcaggcggag gcctggttca agcaggcgga 2820 tcactgagac tgtcatgcgc agcatcaggc cttacatttg atacatatgc aatgggctgg 2880 tttagacaag caccgggaaa aaaacgcgaa tatgttgcag caatttcatg gacaggcatc 2940 agcacatatt atgcggatat tgcaaaagga cgctttacaa ttagccgtga taatgcgaaa 3000 aacacgctgt atctgcaaat ggattcactg aaaccggaag atacagcagt ctattattgc 3060 gcagcacaaa aatcactgaa tgttccggca ccgtgggatt attggggaca aggcacacaa 3120 gttacagttt catcagatcc gtgtccgcaa tgctgcaaac cgtcactgtc actgcaaccg 3180 cctgcactgg cggatctgct gctgggctca aatgcatcac tgacatgcac actgtcagga 3240 ctgaaaaaat ctgaaggcgt ttcatttaca tggcaaccgt caggcggaaa agatgcagtt 3300 caagcatcac cgacaagaga ttcatgcggc tgctattcag tttcaagcat tcttccggga 3360 tgcgctgatc cgtggaataa aggcgaaaca ttttcatgca cagcagcaca ttcagaactg 3420 aaatcagcac tgacagcgac aattacaaaa ccgaaagtca atacatttcg tccgcaagtt 3480 catctgcttc cgcctccgag cgaagaactg gcactgaatg aactggttac acttacatgc 3540 ctggttagag gcttttcacc gaaagatgtt cttgttagat ggctgcaagg cggacaagaa 3600 ctgccgagag ataaatatct ggtttgggaa tcactgccgg aaccgggaca agcaattccg 3660 acatatgcag ttacatcagt tcttagagtt gatgcggaag attggaaaca gggcgataca 3720 tttagctgca tggttggcca tgaagcactg ccgctggcat ttacacaaaa aacaattgat 3780 agactggcag gcaaaccgac acatgttaat gtttcagttg ttatggcaga agcagaaggc 3840 atctgctata aagatgaact gggtggcatg cgtaaaggcg aagagctgtt cactggtttc 3900 gtcactattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc 3960 gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt 4020 aaactgccgg taccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt 4080 gctcgttatc cggaccacat gaagcagcat gacttcttca agtccgccat gccggaaggc 4140 tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa 4200 gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa 4260 gaagacggca atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac 4320 atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg 4380 gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt 4440 cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg 4500 aacgagaaac gcgatcacat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat 4560 ggtatggatg aactgtacaa ataacaccac catcatcacc attaaaagag catgagagga 4620 cggatttcct gaaggaaatc cgttttttta ttttgcccgt cttataaatt tctttgatta 4680 caaagctgtt cttttataat atgagataat gccgactgta ctttttacag tcggttttct 4740 aatgtcacta acctgccccg ttagttgaag aaggttttta tattacagct ccagatcctc 4800 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat 4860 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt 4920 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg 4980 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc 5040 ctaatgcagg agtcgcataa gggagagcgt cgacatggat gagcgatgat gatatccgtt 5100 taggctgggc ggtgatagct tctcgttcag gcagtacgcc tcttttcttt tccagacctg 5160 agggaggcgg aaatggtgtg aggttcccgg ggaaaagcca aataggcgat cgcgggagtg 5220 ctttatttga agatcaggct atcactgcgg tcaatagatt tcacaatgtg atggctggac 5280 agcctgagga actctcgaac ccgaatggaa acaaccagat atttatgaat cagcgcggct 5340 cacatggcgt tgtgctggca aatgcaggtt catcctctgt ctctatcaat acggcaacaa 5400 aattgcctga tggcaggtat gacaataaag ctggagcggg ttcatttcaa gtgaacgatg 5460 gtaaactgac aggcacgatc aatgccaggt ctgtagctgt gctttatcct gatgatattg 5520 caaaagcgcc tcatgttttc cttgagaatt acaaaacagg tgtaacacat tctttcaatg 5580 atcaactgac gattaccttg cgtgcagatg cgaatacaac aaaagccgtt tatcaaatca 5640 ataatggacc agacgacagg cgtttaagga tggagatcaa ttcacaatcg gaaaaggaga 5700 tccaatttgg caaaacatac accatcatgt taaaaggaac gaacagtgat ggtgtaacga 5760 ggaccgagaa atacagtttt gttaaaagag atccagcgtc ggccaaaacc atcggctatc 5820 aaaatccgaa tcattggagc caggtaaatg cttatatcta taaacatgat gggagccgag 5880 taattgaatt gaccggatct tggcctggaa aaccaatgac taaaaatgca gacggaattt 5940 acacgctgac gctgcctgcg gacacggata caaccaacgc aaaagtgatt tttaataatg 6000 gcagcgccca agtgcccggt cagaatcagc ctggctttga ttacgtgcta aatggtttat 6060 ataatgactc gggcttaagc ggttctcttc cccattgagg gcaaggctag acgggactta 6120 ccgaaagaaa ccatcaatga tggtttcttt tttgttcata aatcagacaa aacttttctc 6180 ttgcaaaagt ttgtgaagtg ttgcacaata taaatgtgaa atacttcaca aacaaaaaga 6240 catcaaagag aaacataccc tgcaaggatg ctgatattgt ctgcatttgc gccggagcaa 6300 accaaaaacc tggtgagaca cgccttgaat tagtagaaaa gaacttgaag attttcaaag 6360 gcatcgttag tgaagtcatg gcgagcggat ttgacggcat tttcttagtc gcgacgcgag 6420 gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt 6480 tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc 6540 tcgcggctct taccagccta acttcgatca ctggaccgct gatcgtcacg gcgatttatg 6600 ccgcctcggc gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg 6660 tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctactga agtggatttc 6720 tttaagagct cctttaactt cctcaccagt agttgtatcg gtaccataag tagaagcagc 6780 aacccaagta gctttaccag catccggttc aaccagcata gtaagaatct tactggacat 6840 cggcagttct tcgaacagtg cgccaactac cagctctttc tgcagttcat tcagggcacc 6900 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 6960 tgatccatgg attacgcgtt aacccgggcc cgcggatgca tatgatcaga tcttaaggcc 7020 taggtctaga ggatcgatct gtataataaa gaataattat taatctgtag acaaattgtg 7080 aaaggatgta cttaaacgct aacggtcagc tttattgaac agtaatttaa gtatatgtcc 7140 aatctagggt aagtaaattg agtatcaata taaactttat atgaacataa tcaacgaggt 7200 gaaatcatga gcaatttgat taacggaaaa ataccaaatc aagcgattca aacattaaaa 7260 atcgtaaaag atttatttgg aagttcaata gttggagtat atctatttgg ttcagcagta 7320 aatggtggtt tacgcattaa cagcgatgta gatgttctag tcgtcgtgaa tcatagttta 7380 cctcaattaa ctcgaaaaaa actaacagaa agactaatga ctatatcagg aaagattgga 7440 aatacggatt ctgttagacc acttgaagtt acggttataa ataggagtga agttgtccct 7500 tggcaatatc ctccaaaaag agaatttata tacggtgagt ggctcagggg tgaatttgag 7560 aatggacaaa ttcaggaacc aagctatgat cctgatttgg ctattgtttt agcacaagca 7620 agaaagaata gtatttctct atttggtcct gattcttcaa gtatacttgt ctccgtacct 7680 ttgacagata ttcgaagagc aattaaggat tctttgccag aactaattga ggggataaaa 7740 ggtgatgagc gtaatgtaat tttaacccta gctcgaatgt ggcaaacagt gactactggt 7800 gaaattacct cgaaagatgt cgctgcagaa tgggctatac ctcttttacc taaagagcat 7860 gtaactttac tggatatagc tagaaaaggc tatcggggag agtgtgatga taagtgggaa 7920 ggactatatt caaaggtgaa agcactcgtt aagtatatga aaaattctat agaaacttct 7980 ctcaattagg ctaattttat tgcaataaca ggtgcttact tttctggagt tctttagcaa 8040 atttttttat tagctgaact tagtattagt ggccatactc ctccaatcca aagctattta 8100 gaaagattac tatatcctca aacaggcggt aaccggcctc ttcatcggga atgcgcgcga 8160 ccttcagcat cgccggcatg tccccctggc ggacgggaag tatccagctc gaggtcgggc 8220 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 8280 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 8340 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 8400 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 8460 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 8520 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 8580 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 8640 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 8700 gctgaagcca gttaccttcg gaaaaagagt tgatagctct tgatccggca aacaaaccac 8760 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 8820 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 8880 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 8940 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 9000 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 9060 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 9120 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 9180 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 9240 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 9300 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 9360 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 9420 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 9480 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 9540 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 9600 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 9660 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 9720 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 9780 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 9840 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 9900 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 9960 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 10020 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaa 10063

<210> 223

<211> 20

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (20)

<223> primer E257

<400> 223 ctgagtcaat ttcatcaagg 20

<210> 224

<211> 22

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (22 )

<223> priomer E258

<400> 224 cagaagaaaa cattctcgaa gc 22

<210> 225

<211> 20

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (20)

<223> primer E261

<400> 225 gaggtcgcaa ctgaaaaagc 20

<210> 226

<211> 20

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (20)

<223> primer E262

<400> 226 atgttcagtc tccttactcc 20 <210> 227

<211> 30

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (30)

<223> primer E372

<400> 227 attataacau gtattcacga acgaaaatcg 30

<210> 228

<211> 35

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (35)

<223> primer E373

<400> 228 atcagatttu agaaaacaat aaacccttgc atagg 35

<210> 229

<211> 20

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (20)

<223> primer E374

<400> 229 aaaatctgau acggggcagg 20

<210> 230

<211> 37

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (37 )

<223> primer E375

<400> 230 atgttataau acccattagt tcaacaaacg aaaattg 37

<210> 231

<211> 264 <212> PRT

<213> synthetic

<220>

<221> PEPTIDE

<222> (1) . . (264 )

<223> KanR

<400> 231

Met Ile Glu Gin Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gin Gin Thr Ile Gly Cys Ser 20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gin Gly Arg Pro Val Leu Phe 35 40 45 Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gin Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val 65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95 Val Pro Gly Gin Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110 Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro 115 120 125

Ala Thr Cys Pro Phe Asp His Gin Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gin Asp Asp Leu Asp Glu 145 150 155 160

Glu His Gin Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys 180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp 195 200 205 Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gin Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe 225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gin Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe 260

<210> 232

<211> 2734

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (2734 )

<223> Intcgration fragment INTI comprising P (gsh2 ) promoter, cial essential gene, and TIS1 translation initiation sequence .

<220>

<221> CDS

<222> (1024 ) . . (1398)

<223> blaShe (resistance gene used to select for chromosomal intcgration)

<220>

<221> promoter

<222> (1724 ) . . (2234 )

<223> P13_pGSH2

<220>

<221> promoter

<222> (2229) . . (2234 )

<223> TIS1

<220>

<221> gene

<222> (2235) . . (2734 )

<223> first part of CIA1 gene, for intcgration by homologous recombination

<400> 232 tggtcctcgt ctatttgttg tttaaaagtg atagtatact tcttgaactc gctggggtta 60 agaaattcca agtcttcatc ttctacaaag aagtagttgt acgagaacaa t agccat aca 120 gtaccaccta tgacgaccag actaggaata aatctcaacc atgggtttct ccgctttcta 180 atgacctttt ctgctttcaa ctccgatggt gaaggtggag gctggtgttc ataagtgacc 240 ctttggttgg gtcttggaat gtattgtttt ttcagttgaa tcccttgcaa eggttgaagg 300 ccgccagtca acttgggggc gtttttctta gcagggactg cataatttct aacgagcegt 360 agtattctet ggttcatgga tcatagaact gaggggaagg ggaagtgtag aaatgagcag 420 aaatggtagg ggatttggag ttctetctaa gcagcgaaca gaaaattcaa gcaccctaga 480 agcagatccc attaatgeaa tccgccccat caaccatcaa ctacaagatc tatcacaaac 540 acacccacac accatagctt caaaatgttt ctactccttt tttactcttc cagattttct 600 cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc 660 ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag 720 agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct 780 ttttcttgaa attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa 840 gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac 900 tttttttact tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt 960 gacaattaat catcggcata gtatatcgge atagtataat acgacaaggt gaggaactaa 1020 acc atg gcc aag ttg acc agt gcc gtt ccg gtg ctc acc gcg cgc gac 1068 Met Ala Lys Leu Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp 1 5 10 15 gtc gct gga gcg gtc gag ttc tgg acc gac egg ctc ggg ttc tcc egg 1116 Val Ala Gly Ala Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg 20 25 30 gac ttc gtg gag gac gac ttc gct ggt gtg gtc egg gac gac gtg acc 1164 Asp Phe Val Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr 35 40 45 ctg ttc atc agc gcg gtc cag gac cag gtg gtg ccg gac aac acc ttg 1212 Leu Phe Ile Ser Ala Val Gin Asp Gin Val Val Pro Asp Asn Thr Leu 50 55 60 gct tgg gtg tgg gtg cgc ggt ttg gac gag ctg tac gcc gag tgg tcg 1260 Ala Tip Val Tip Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser 65 70 75 gag gtc gtg tcc acg aac ttc egg gac gcc tcc ggt cca gcc atg acc 1308 Glu Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr 80 85 90 95 gag atc ggc gag cag ccg tgg ggg agg gag ttc gcc ctg cgc gat ccg 1356 Glu Ile Gly Glu Gin Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro 100 105 110 gct ggc aac tgc gtt cac ttc gtg gcc gag gag cag gac tga 1398 Ala Gly Asn Cys Val His Phe Val Ala Glu Glu Gin Asp 115 120 cacgtccgac ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt 1458 cgatatcatg taattagtta tgtcaegctt acattcacgc cctcccccca catccgctct 1518 aaccgaaaag gaaggagtta gacaacctga agtctaggte cctatttatt tttttatagt 1578 tatgttagta ttaagaaegt tatttatatt tcaaattttt cttttttttc tgtacagacg 1638 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gaegctcgaa 1698 ggctttaatt tgcaagctat caactctggc ccgaggaagt cagcgaaaga ggatacttta 1758 cccccagagg agaatataaa attgatgaca atgcagctca agccatgaaa gactcaatgc 1818 tgtacaagat gtcattttac agattcggtg aacttttccc ctctggagat gccatagacc 1878 gtgtcagagg tcaaagattg agtcgctctt acgctgagtc tatagatctt aacattgttg 1938 aagaagtatt cacaagtgag aactggctgg ttcgtcttta caagctcaaa gagccagaca 1998 acttgggaag aagcctactg accctgaagg ataacgagaa aaagttggcc actaaaaaag 2058 gtcgcagatt gagggtcaac aagaaaccct cattggatct acgggtgtag tgtagttgtg 2118 tattttagat agacagagct ctggggttag aatgataaca tattgcgtgc ttatgtaatg 2178 atgttcacct ttcgtaggtc tcaaaagtta gcctagtctt agcccgtaac tgatatatgt 2238 ctctatcttt ggttcacaca ttcacaggtc acacagataa atgctgggct acttccattc 2298 actctaaact tcctttattg gcaacagtct ccggggacag aacttgtcgg gtctataact 2358 tggaaacgaa gagactgatt gtcgtactcg acgatgattc tcattcaaag actctttcca 2418 gcgtcgaatg gaagccaact ggggaattcc cttcgttggc aatcggctcg tttgattcta 2478 caatctctat ttgggggaat gaagaagctg tcctggaaga cgaggataac tggacactga 2538 tggccattat cgaaggacat gagaacgaga ttaaaggggt gagttggagc catgatggag 2598 tgtatctggc atcatgctca cgagacaagt ccatttggat atgggaggct gatgataaca 2658 acgaagagtt tgagtgcgtc tttgttgtac aggaacacag ccaagatgtc aagcatgtaa 2718 catggcatca gcatga 2734

<210> 233

<211> 124

<212> PRT

<213> synthetic

<400> 233

Met Ala Lys Leu Thr Ser Ala Val Pro Val Leu Thr Ala Arg Asp Val 1 5 10 15

Ala Gly Ala Val Glu Phe Trp Thr Asp Arg Leu Gly Phe Ser Arg Asp 20 25 30

Phe Val Glu Asp Asp Phe Ala Gly Val Val Arg Asp Asp Val Thr Leu 35 40 45

Phe Ile Ser Ala Val Gin Asp Gin Val Val Pro Asp Asn Thr Leu Ala 50 55 60

Trp Val Trp Val Arg Gly Leu Asp Glu Leu Tyr Ala Glu Trp Ser Glu 65 70 75 80 Val Val Ser Thr Asn Phe Arg Asp Ala Ser Gly Pro Ala Met Thr Glu 85 90 95 Ile Gly Glu Gin Pro Trp Gly Arg Glu Phe Ala Leu Arg Asp Pro Ala 100 105 110

Gly Asn Cys Val His Phe Val Ala Glu Glu Gin Asp 115 120

<210> 234

<211> 2734

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (2734 )

<223> Intcgration fragment INT3 comprising P (gsh2 ) promoter, cial essential gene, and TIS3 translation initiation sequence .

<220>

<221> promoter

<222> (1724 ) . . (2234 )

<223> P13_pGSH2

<220>

<221> promoter

<222> (2229) . . (2234 )

<223> TIS3

<220>

<221> gene

<222> (2235) . . (2734 )

<223> first part of CIA1 gene, for intcgration by homologous recombination

<400> 234 tggtcctcgt ctatttgttg tttaaaagtg atagtatact tcttgaactc gctggggtta 60 agaaattcca agtcttcatc ttctacaaag aagtagttgt acgagaacaa tagccataca 120 gtaccaccta tgacgaccag actaggaata aatctcaacc atgggtttct ccgctttcta 180 atgacctttt ctgctttcaa ctccgatggt gaaggtggag gctggtgttc ataagtgacc 240 ctttggttgg gtcttggaat gtattgtttt ttcagttgaa tcccttgcaa cggttgaagg 300 ccgccagtca acttgggggc gtttttctta gcagggactg cataatttct aacgagccgt 360 agtattctct ggttcatgga tcatagaact gaggggaagg ggaagtgtag aaatgagcag 420 aaatggtagg ggatttggag ttctctctaa gcagcgaaca gaaaattcaa gcaccctaga 480 agcagatccc attaatgcaa tccgccccat caaccatcaa ctacaagatc tatcacaaac 540 acacccacac accatagctt caaaatgttt ctactccttt tttactcttc cagattttct 600 cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc 660 ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag 720 agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct 780 ttttcttgaa attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa 840 gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac 900 tttttttact tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt 960 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa 1020 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgctggagcg 1080 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgct 1140 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 1200 gacaacacct tggcttgggt gtgggtgcgc ggtttggacg agctgtacgc cgagtggtcg 1260 gaggtcgtgt ccacgaactt ccgggacgcc tccggtccag ccatgaccga gatcggcgag 1320 cagccgtggg ggagggagtt cgccctgcgc gatccggctg gcaactgcgt tcacttcgtg 1380 gccgaggagc aggactgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc 1440 gtcccccttt tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc 1500 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc 1560 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct 1620 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa 1680 ggttttggga cgctcgaagg ctttaatttg caagctatca actctggccc gaggaagtca 1740 gcgaaagagg atactttacc cccagaggag aatataaaat tgatgacaat gcagctcaag 1800 ccatgaaaga ctcaatgctg tacaagatgt cattttacag attcggtgaa cttttcccct 1860 ctggagatgc catagaccgt gtcagaggtc aaagattgag tcgctcttac gctgagtcta 1920 tagatcttaa cattgttgaa gaagtattca caagtgagaa ctggctggtt cgtctttaca 1980 agctcaaaga gccagacaac ttgggaagaa gcctactgac cctgaaggat aacgagaaaa 2040 agttggccac taaaaaaggt cgcagattga gggtcaacaa gaaaccctca ttggatctac 2100 gggtgtagtg tagttgtgta ttttagatag acagagctct ggggttagaa tgataacata 2160 ttgcgtgctt atgtaatgat gttcaccttt cgtaggtctc aaaagttagc ctagtcttag 2220 cccgtaacac gttcatgtct ctatctttgg ttcacacatt cacaggtcac acagataaat 2280 gctgggctac ttccattcac tctaaacttc ctttattggc aacagtctcc ggggacagaa 2340 cttgtcgggt ctataacttg gaaacgaaga gactgattgt cgtactcgac gatgattctc 2400 attcaaagac tctttccagc gtcgaatgga agccaactgg ggaattccct tcgttggcaa 2460 tcggctcgtt tgattctaca atctctattt gggggaatga agaagctgtc ctggaagacg 2520 aggataactg gacactgatg gccattatcg aaggacatga gaacgagatt aaaggggtga 2580 gttggagcca tgatggagtg tatctggcat catgctcacg agacaagtcc atttggatat 2640 gggaggctga tgataacaac gaagagtttg agtgcgtctt tgttgtacag gaacacagcc 2700 aagatgtcaa gcatgtaaca tggcatcagc atga 2734

<210> 235

<211> 2734

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (2734 )

<223> Intcgration fragment INT5 comprising P (gsh2 ) promoter, cial essential gene, and TIS5 translation initiation sequence .

<220>

<221> promoter

<222> (1724 ) . . (2234 )

<223> P13_pGSH2

<220>

<221> promoter

<222> (2229) . . (2234 )

<223> TIS5

<220>

<221> gene

<222> (2235) . . (2734 )

<223> first part of CIA1 gene, for intcgration by homologous recombination

<400> 235 tggtcctcgt ctatttgttg tttaaaagtg atagtatact tcttgaactc gctggggtta 60 agaaattcca agtcttcatc ttctacaaag aagtagttgt acgagaacaa tagccataca 120 gtaccaccta tgacgaccag actaggaata aatctcaacc atgggtttct ccgctttcta 180 atgacctttt ctgctttcaa ctccgatggt gaaggtggag gctggtgttc ataagtgacc 240 ctttggttgg gtcttggaat gtattgtttt ttcagttgaa tcccttgcaa cggttgaagg 300 ccgccagtca acttgggggc gtttttctta gcagggactg cataatttct aacgagccgt 360 agtattctct ggttcatgga tcatagaact gaggggaagg ggaagtgtag aaatgagcag 420 aaatggtagg ggatttggag ttctctctaa gcagcgaaca gaaaattcaa gcaccctaga 480 agcagatccc attaatgcaa tccgccccat caaccatcaa ctacaagatc tatcacaaac 540 acacccacac accatagctt caaaatgttt ctactccttt tttactcttc cagattttct 600 cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc 660 ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag 720 agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct 780 ttttcttgaa attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa 840 gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac 900 tttttttact tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt 960 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa 1020 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgctggagcg 1080 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgct 1140 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 1200 gacaacacct tggcttgggt gtgggtgcgc ggtttggacg agctgtacgc cgagtggtcg 1260 gaggtcgtgt ccacgaactt ccgggacgcc tccggtccag ccatgaccga gatcggcgag 1320 cagccgtggg ggagggagtt cgccctgcgc gatccggctg gcaactgcgt tcacttcgtg 1380 gccgaggagc aggactgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc 1440 gtcccccttt tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc 1500 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc 1560 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct 1620 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa 1680 ggttttggga cgctcgaagg ctttaatttg caagctatca actctggccc gaggaagtca 1740 gcgaaagagg atactttacc cccagaggag aatataaaat tgatgacaat gcagctcaag 1800 ccatgaaaga ctcaatgctg tacaagatgt cattttacag attcggtgaa cttttcccct 1860 ctggagatgc catagaccgt gtcagaggtc aaagattgag tcgctcttac gctgagtcta 1920 tagatcttaa cattgttgaa gaagtattca caagtgagaa ctggctggtt cgtctttaca 1980 agctcaaaga gccagacaac ttgggaagaa gcctactgac cctgaaggat aacgagaaaa 2040 agttggccac taaaaaaggt cgcagattga gggtcaacaa gaaaccctca ttggatctac 2100 gggtgtagtg tagttgtgta ttttagatag acagagctct ggggttagaa tgataacata 2160 ttgcgtgctt atgtaatgat gttcaccttt cgtaggtctc aaaagttagc ctagtcttag 2220 cccgtaacta ggttatgtct ctatctttgg ttcacacatt cacaggtcac acagataaat 2280 gctgggctac ttccattcac tctaaacttc ctttattggc aacagtctcc ggggacagaa 2340 cttgtcgggt ctataacttg gaaacgaaga gactgattgt cgtactcgac gatgattctc 2400 attcaaagac tctttccagc gtcgaatgga agccaactgg ggaattccct tcgttggcaa 2460 tcggctcgtt tgattctaca atctctattt gggggaatga agaagctgtc ctggaagacg 2520 aggataactg gacactgatg gccattatcg aaggacatga gaacgagatt aaaggggtga 2580 gttggagcca tgatggagtg tatctggcat catgctcacg agacaagtcc atttggatat 2640 gggaggctga tgataacaac gaagagtttg agtgcgtctt tgttgtacag gaacacagcc 2700 aagatgtcaa gcatgtaaca tggcatcagc atga 2734

<210> 236

<211> 2734

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1) . . (2734 )

<223> Intcgration fragment INT9 comprising P (gsh2 ) promoter, cial essential gene, and TIS9 translation initiation sequence .

<220>

<221> promoter

<222> (1724 ) . . (2234 )

<223> P13_pGSH

<220>

<221> promoter

<222> (2229) . . (2234 )

<223> TIS9

<220>

<221> gene

<222> (2235) . . (2734 )

<223> first part of CIA1 gene, for intcgration by homologous recombination

<400> 236 tggtcctcgt ctatttgttg tttaaaagtg atagtatact tcttgaactc gctggggtta 60 agaaattcca agtcttcatc ttctacaaag aagtagttgt acgagaacaa tagccataca 120 gtaccaccta tgacgaccag actaggaata aatctcaacc atgggtttct ccgctttcta 180 atgacctttt ctgctttcaa ctccgatggt gaaggtggag gctggtgttc ataagtgacc 240 ctttggttgg gtcttggaat gtattgtttt ttcagttgaa tcccttgcaa cggttgaagg 300 ccgccagtca acttgggggc gtttttctta gcagggactg cataatttct aacgagccgt 360 agtattctct ggttcatgga tcatagaact gaggggaagg ggaagtgtag aaatgagcag 420 aaatggtagg ggatttggag ttctctctaa gcagcgaaca gaaaattcaa gcaccctaga 480 agcagatccc attaatgcaa tccgccccat caaccatcaa ctacaagatc tatcacaaac 540 acacccacac accatagctt caaaatgttt ctactccttt tttactcttc cagattttct 600 cggactccgc gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc 660 ctctttcttc ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag 720 agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct 780 ttttcttgaa attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa 840 gttaataaac ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac 900 tttttttact tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt 960 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa 1020 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgctggagcg 1080 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgct 1140 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 1200 gacaacacct tggcttgggt gtgggtgcgc ggtttggacg agctgtacgc cgagtggtcg 1260 gaggtcgtgt ccacgaactt ccgggacgcc tccggtccag ccatgaccga gatcggcgag 1320 cagccgtggg ggagggagtt cgccctgcgc gatccggctg gcaactgcgt tcacttcgtg 1380 gccgaggagc aggactgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc 1440 gtcccccttt tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc 1500 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc 1560 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct 1620 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa 1680 ggttttggga cgctcgaagg ctttaatttg caagctatca actctggccc gaggaagtca 1740 gcgaaagagg atactttacc cccagaggag aatataaaat tgatgacaat gcagctcaag 1800 ccatgaaaga ctcaatgctg tacaagatgt cattttacag attcggtgaa cttttcccct 1860 ctggagatgc catagaccgt gtcagaggtc aaagattgag tcgctcttac gctgagtcta 1920 tagatcttaa cattgttgaa gaagtattca caagtgagaa ctggctggtt cgtctttaca 1980 agctcaaaga gccagacaac ttgggaagaa gcctactgac cctgaaggat aacgagaaaa 2040 agttggccac taaaaaaggt cgcagattga gggtcaacaa gaaaccctca ttggatctac 2100 gggtgtagtg tagttgtgta ttttagatag acagagctct ggggttagaa tgataacata 2160 ttgcgtgctt atgtaatgat gttcaccttt cgtaggtctc aaaagttagc ctagtcttag 2220 cccgtaacaa aacaatgtct ctatctttgg ttcacacatt cacaggtcac acagataaat 2280 gctgggctac ttccattcac tctaaacttc ctttattggc aacagtctcc ggggacagaa 2340 cttgtcgggt ctataacttg gaaacgaaga gactgattgt cgtactcgac gatgattctc 2400 attcaaagac tctttccagc gtcgaatgga agccaactgg ggaattccct tcgttggcaa 2460 tcggctcgtt tgattctaca atctctattt gggggaatga agaagctgtc ctggaagacg 2520 aggataactg gacactgatg gccattatcg aaggacatga gaacgagatt aaaggggtga 2580 gttggagcca tgatggagtg tatctggcat catgctcacg agacaagtcc atttggatat 2640 gggaggctga tgataacaac gaagagtttg agtgcgtctt tgttgtacag gaacacagcc 2700 aagatgtcaa gcatgtaaca tggcatcagc atga 2734 <210> 237

<211> 19

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (19)

<223> primer E251

<400> 237 acgcgtgtac gcatgtaac 19

<210> 238

<211> 20

<212> DNA

<213> synthetic

<220>

<221> primer_bind

<222> (1) • • (20)

<223> primer E627

<400> 238 cataactgga gcttgctagg 20

<210> 239

<211> 750

<212> DNA

<213> Aspergillus oryzae

<220>

<221> promoter

<222> (1) • • (750)

<223> Aspergillus oryzae burden sensing promoter p (agsA)

<400> 239 gttgtttctt aatataggca cacggatccc gccatcgatc ggcgccctgt tcgagtatgg 60 ccatcctcac acattgtcgg gctccgctgg cgctgcctca acatattttt aaatggggaa 120 accataagca aagagactcc atcgggcgca ctacacatct cgcctcttct ggaccctgtg 180 gatcgggact ggggtggatg gcaaaccgtc aagttgccgc cgcttcttcg cgctgttgcg 240 gcgtcacgcc atataggcga ctctcccaat catggaaagc ctgcgttcca gcgatattca 300 acgacgtggt ggcttggatt gcagcttcga ccagatcaca gcaatagaat agcgatgatt 360 ccagccgtgg gcctttaggg tagaaaccat actgggctag gaaacatgcc gtatcaagtg 420 agaaacaccg ctaccccacc acggctgaaa agtagatagt ggatggttcc atgtcatcta 480 tcagcaggcg aggagtgggt ctatagtaga ggttgtaatg gtcgtggtag aggtccctgg 540 cccattttta tccccatcct tcccggcctg atggttccaa gccttcttta cgaaaagttt 600 tgcagctgct ttttcgagtc gacaggtcgc tcgttccttc gtttgatttc cctcgctttt 660 gaccacgccc agggcatgta ggatttattc cgtttgagat tatcgagagc ctaagtcgcg 720 cgcagttgtc ggtagcaggc cactgccggg 750

<210> 240

<211> 1200

<212> DNA

<213> Aspergillus oryzae

<220>

<221> CDS

<222> (1) . . (1200)

<223> ERG10 Aspergillus essential gene (mRNA)

<400> 240 atg tct tct ctt ccg ccg gtc tat att gtt tcc tct gcc cgc acc cca 48

Met Ser Ser Leu Pro Pro Val Tyr Ile Val Ser Ser Ala Arg Thr Pro 1 5 10 15 gtc ggc tct ttc ttg ggg tcg ctc tca agt ctc act gcc ccg cag tta 96 Val Gly Ser Phe Leu Gly Ser Leu Ser Ser Leu Thr Ala Pro Gin Leu

20 25 30 ggc tct cat gct att aaa gct gcg ctc agc aaa gcg gat gga atc aag 144

Gly Ser His Ala Ile Lys Ala Ala Leu Ser Lys Ala Asp Gly Ile Lys

35 40 45 ccg tct gat atc cag gag gtc ttc ttt ggc aat gtc atc tcc gca aac 192

Pro Ser Asp Ile Gin Glu Val Phe Phe Gly Asn Val Ile Ser Ala Asn

50 55 60 gtt gga caa aat cct gct aga cag tgt gct ctc ggc gct ggt ctc aat 240 Val Gly Gin Asn Pro Ala Arg Gin Cys Ala Leu Gly Ala Gly Leu Asn

65 70 75 80 gaa tca act gtc tgt act acg gtt aat aag gtg tgc gcg tct ggc ttg 288

Glu Ser Thr Val Cys Thr Thr Val Asn Lys Val Cys Ala Ser Gly Leu

85 90 95 aaa gcg gtt att ctc ggt gca cag acc atc atg act ggc aat gcg gat 336

Lys Ala Val Ile Leu Gly Ala Gin Thr Ile Met Thr Gly Asn Ala Asp

100 105 110 att gtc gta gca ggc ggt gct gaa tcc atg tct aac gcc cct cat tac 384 Ile Val Val Ala Gly Gly Ala Glu Ser Met Ser Asn Ala Pro His Tyr

115 120 125 ctt cca aac ctt cgc gtc ggt gcg aaa tac ggc aac cag agt ctg gtg 432

Leu Pro Asn Leu Arg Val Gly Ala Lys Tyr Gly Asn Gin Ser Leu Val

130 135 140 gac ggt att atg aag gat ggc ttg aca gac gca gga aag cag gaa ctc 480

Asp Gly Ile Met Lys Asp Gly Leu Thr Asp Ala Gly Lys Gin Glu Leu

145 150 155 160 atg ggc ttg caa gcc gag gag tgt gct cag gat cat ggc ttt agc agg 528

Met Gly Leu Gin Ala Glu Glu Cys Ala Gin Asp His Gly Phe Ser Arg

165 170 175 gaa caa cag gat gat tat gcc att cgc act tac gaa aaa gca cag gcg 576

Glu Gin Gin Asp Asp Tyr Ala Ile Arg Thr Tyr Glu Lys Ala Gin Ala

180 185 190 gct caa aag gct ggc ctt ttt gac gaa gaa att gcg cct att gaa ctt 624

Ala Gin Lys Ala Gly Leu Phe Asp Glu Glu Ile Ala Pro Ile Glu Leu

195 200 205 cct ggc ttt agg ggc aag cca ggt gtg act gtg tca caa gac gaa gaa 672

Pro Gly Phe Arg Gly Lys Pro Gly Val Thr Val Ser Gin Asp Glu Glu

210 215 220 cca aag aat ctt aac ccg gat aag ctt cga gct atc aag cct gca ttt 720

Pro Lys Asn Leu Asn Pro Asp Lys Leu Arg Ala Ile Lys Pro Ala Phe

225 230 235 240 atc ccc gga tcc ggc acg gtc aca gcc ccg aat tcc tca cct ctt aac 768 Ile Pro Gly Ser Gly Thr Val Thr Ala Pro Asn Ser Ser Pro Leu Asn

245 250 255 gac ggt gct gct gct gtt atc ctc gtc tca gaa gct aaa ctg aaa gag 816

Asp Gly Ala Ala Ala Val Ile Leu Val Ser Glu Ala Lys Leu Lys Glu

260 265 270 ctt aac cta aag cct gtt gca aag att ctt ggc tgg gga gat gcc gcc 864

Leu Asn Leu Lys Pro Val Ala Lys Ile Leu Gly Trp Gly Asp Ala Ala

275 280 285 cag cag cca agc aaa ttc aca act gcc cca gct cta gca att ccc aag 912

Gin Gin Pro Ser Lys Phe Thr Thr Ala Pro Ala Leu Ala Ile Pro Lys

290 295 300 gcc ctc agc cat gca ggt gtg gct cag gat gct gtt gat gcg ttc gag 960

Ala Leu Ser His Ala Gly Val Ala Gin Asp Ala Val Asp Ala Phe Glu

305 310 315 320 att aac gaa gcg ttc agc gta gtt gct ctg gcc aat atg aaa ctc ctg 1008 Ile Asn Glu Ala Phe Ser Val Val Ala Leu Ala Asn Met Lys Leu Leu

325 330 335 ggg ttg gct gaa gat aaa gtc aac atc cat ggt ggt gca gtg gct atc 1056

Gly Leu Ala Glu Asp Lys Val Asn Ile His Gly Gly Ala Val Ala Ile

340 345 350 ggt cat cct atc ggc gcc agc ggt gct cgt atc ttg act aca ttg ctc 1104

Gly His Pro Ile Gly Ala Ser Gly Ala Arg Ile Leu Thr Thr Leu Leu

355 360 365 ggt gta ttg aaa gcg aga aag ggt aag att ggt tgt gcc ggg att tgt 1152

Gly Val Leu Lys Ala Arg Lys Gly Lys Ile Gly Cys Ala Gly Ile Cys

370 375 380 aat gga gga ggt ggt gct agc gct att gtt gtc gaa tct ctc gtc tga 1200

Asn Gly Gly Gly Gly Ala Ser Ala Ile Val Val Glu Ser Leu Val

385 390 395

<210> 241

<211> 399

<212> PRT

<213> Aspergillus oryzae <400> 241

Met Ser Ser Leu Pro Pro Val Tyr Ile Val Ser Ser Ala Arg Thr Pro

1 5 10 15 Val Gly Ser Phe Leu Gly Ser Leu Ser Ser Leu Thr Ala Pro Gin Leu 20 25 30

Gly Ser His Ala Ile Lys Ala Ala Leu Ser Lys Ala Asp Gly Ile Lys 35 40 45

Pro Ser Asp Ile Gin Glu Val Phe Phe Gly Asn Val Ile Ser Ala Asn 50 55 60 Val Gly Gin Asn Pro Ala Arg Gin Cys Ala Leu Gly Ala Gly Leu Asn 65 70 75 80

Glu Ser Thr Val Cys Thr Thr Val Asn Lys Val Cys Ala Ser Gly Leu 85 90 95

Lys Ala Val Ile Leu Gly Ala Gin Thr Ile Met Thr Gly Asn Ala Asp 100 105 110 Ile Val Val Ala Gly Gly Ala Glu Ser Met Ser Asn Ala Pro His Tyr

115 120 125

Leu Pro Asn Leu Arg Val Gly Ala Lys Tyr Gly Asn Gin Ser Leu Val

130 135 140

Asp Gly Ile Met Lys Asp Gly Leu Thr Asp Ala Gly Lys Gin Glu Leu 145 150 155 160

Met Gly Leu Gin Ala Glu Glu Cys Ala Gin Asp His Gly Phe Ser Arg

165 170 175

Glu Gin Gin Asp Asp Tyr Ala Ile Arg Thr Tyr Glu Lys Ala Gin Ala 180 185 190

Ala Gin Lys Ala Gly Leu Phe Asp Glu Glu Ile Ala Pro Ile Glu Leu 195 200 205

Pro Gly Phe Arg Gly Lys Pro Gly Val Thr Val Ser Gin Asp Glu Glu

210 215 220

Pro Lys Asn Leu Asn Pro Asp Lys Leu Arg Ala Ile Lys Pro Ala Phe 225 230 235 240 Ile Pro Gly Ser Gly Thr Val Thr Ala Pro Asn Ser Ser Pro Leu Asn 245 250 255

Asp Gly Ala Ala Ala Val Ile Leu Val Ser Glu Ala Lys Leu Lys Glu 260 265 270

Leu Asn Leu Lys Pro Val Ala Lys Ile Leu Gly Trp Gly Asp Ala Ala 275 280 285

Gin Gin Pro Ser Lys Phe Thr Thr Ala Pro Ala Leu Ala Ile Pro Lys

290 295 300

Ala Leu Ser His Ala Gly Val Ala Gin Asp Ala Val Asp Ala Phe Glu 305 310 315 320 Ile Asn Glu Ala Phe Ser Val Val Ala Leu Ala Asn Met Lys Leu Leu

325 330 335

Gly Leu Ala Glu Asp Lys Val Asn Ile His Gly Gly Ala Val Ala Ile 340 345 350

Gly His Pro Ile Gly Ala Ser Gly Ala Arg Ile Leu Thr Thr Leu Leu 355 360 365

Gly Val Leu Lys Ala Arg Lys Gly Lys Ile Gly Cys Ala Gly Ile Cys

370 375 380

Asn Gly Gly Gly Gly Ala Ser Ala Ile Val Val Glu Ser Leu Val 385 390 395

<210> 242

<211> 2082

<212> DNA

<213> Aspergillus oryzae

<220>

<221> CDS

<222> (1) . . (2082 )

<223> PFS2 Aspergillus essential gene (mRNA)

<400> 242 atg tgc ggc atc ttc ggc tac att aac tac ctc gtc gag agg gac cgc 48 Met Cys Gly Ile Phe Gly Tyr Ile Asn Tyr Leu Val Glu Arg Asp Arg 1 5 10 15 aag ttt att ctt gac act ctt ctc aat gga ctc tec cgt ctt gaa tac 96 Lys Phe Ile Leu Asp Thr Leu Leu Asn Gly Leu Ser Arg Leu Glu Tyr 20 25 30 cga ggc tac gac tct gcg ggt ctt gcc ata gat ggc gat aag aag aat 144 Arg Gly Tyr Asp Ser Ala Gly Leu Ala Ile Asp Gly Asp Lys Lys Asn 35 40 45 gaa gtt tgt gct ttc aag gaa gtg ggc aag gtt gct aag ctt cga gag 192 Glu Val Cys Ala Phe Lys Glu Val Gly Lys Val Ala Lys Leu Arg Glu 50 55 60 ctt att gat gag tgc aag cct gac ttg acc aaa tcc ttt gaa tct cat 240

Leu Ile Asp Glu Cys Lys Pro Asp Leu Thr Lys Ser Phe Glu Ser His

65 70 75 80 gct ggt att tct cac act cgt tgg gcc act cat ggg acg cct tcc cgc 288

Ala Gly Ile Ser His Thr Arg Trp Ala Thr His Gly Thr Pro Ser Arg

85 90 95 ttg aac tgc cac ccc cac agg tcc gat ctg aac tgg gaa ttt tca gtt 336

Leu Asn Cys His Pro His Arg Ser Asp Leu Asn Trp Glu Phe Ser Val

100 105 110 gtc cac aat ggt atc att acc aac tac aaa gag ctc aag gct ttg ttg 384 Val His Asn Gly Ile Ile Thr Asn Tyr Lys Glu Leu Lys Ala Leu Leu

115 120 125 gag agc aag gga ttc cgc ttt gag act gac aca gac acc gaa tgt att 432

Glu Ser Lys Gly Phe Arg Phe Glu Thr Asp Thr Asp Thr Glu Cys Ile

130 135 140 gca aag ctt acc aaa tat ctc tat gac caa cag ccc gat atc gaa ttc 480

Ala Lys Leu Thr Lys Tyr Leu Tyr Asp Gin Gin Pro Asp Ile Glu Phe

145 150 155 160 aca gtt ttg gct aag gct gtt gtg aag gag ctt gag ggc gcg ttt ggg 528

Thr Val Leu Ala Lys Ala Val Val Lys Glu Leu Glu Gly Ala Phe Gly

165 170 175 ctt ctc atc aaa tcc gta cat tat ccc cat gag gtc att gcg gct cgc 576

Leu Leu Ile Lys Ser Val His Tyr Pro His Glu Val Ile Ala Ala Arg

180 185 190 aag ggg tcg ccg ctt gtt att ggc gtg aga acg tcg aag aag atg aag 624

Lys Gly Ser Pro Leu Val Ile Gly Val Arg Thr Ser Lys Lys Met Lys

195 200 205 gtg gat ttc gtg gac gtc gaa tac tca gaa gat gga gcc ctt cct gca 672 Val Asp Phe Val Asp Val Glu Tyr Ser Glu Asp Gly Ala Leu Pro Ala

210 215 220 gag cag gcc tcc cag aac gtg gcc atc aag aag tct gcg acc ggc ctc 720

Glu Gin Ala Ser Gin Asn Val Ala Ile Lys Lys Ser Ala Thr Gly Leu

225 230 235 240 ctt gcc cca cct gac aaa tcc ctt ttg cat agg tca cag tct cgc gct 768

Leu Ala Pro Pro Asp Lys Ser Leu Leu His Arg Ser Gin Ser Arg Ala

245 250 255 ttc ctt tcc gat gat ggc atc ccc cag cca gct gag ttt ttc ttg tct 816

Phe Leu Ser Asp Asp Gly Ile Pro Gin Pro Ala Glu Phe Phe Leu Ser

260 265 270 tct gac cca tct gca att gtc gag cat acg aag aag gtc ctc tat ctc 864

Ser Asp Pro Ser Ala Ile Val Glu His Thr Lys Lys Val Leu Tyr Leu

275 280 285 gaa gat gat gat atc gct cat gtg cat gag gga cag tta aac att cat 912

Glu Asp Asp Asp Ile Ala His Val His Glu Gly Gin Leu Asn Ile His

290 295 300 cgc ctc aca aaa gat gat ggt act tct aat gtt cgc gct att cag aca 960 Arg Leu Thr Lys Asp Asp Gly Thr Ser Asn Val Arg Ala Ile Gin Thr 305 310 315 320 att gaa ctc gaa ttg cag gag att atg aag ggc aag ttt gac cac ttc 1008 Ile Glu Leu Glu Leu Gin Glu Ile Met Lys Gly Lys Phe Asp His Phe

325 330 335 atg caa aag gaa att ttc gag caa ccc gag tct gtg atc aat acc atg 1056

Met Gin Lys Glu Ile Phe Glu Gin Pro Glu Ser Val Ile Asn Thr Met

340 345 350 aga gga cga ctg gat gtt gca aac aag caa gtc aca ctc ggt ggc ctg 1104

Arg Gly Arg Leu Asp Val Ala Asn Lys Gin Val Thr Leu Gly Gly Leu

355 360 365 cgg cag tac att tct act atc cgc cgc tgc aga aga atc ata ttt gtt 1152

Arg Gin Tyr Ile Ser Thr Ile Arg Arg Cys Arg Arg Ile Ile Phe Val

370 375 380 gcc tgt gga act agt tac cat tca tgc atg gct gtg cgt gga gtc ttt 1200

Ala Cys Gly Thr Ser Tyr His Ser Cys Met Ala Val Arg Gly Val Phe

385 390 395 400 gaa gag ctt act gag att cct att tct gtt gaa ctt gct tcc gat ttc 1248

Glu Glu Leu Thr Glu Ile Pro Ile Ser Val Glu Leu Ala Ser Asp Phe

405 410 415 ctg gat aga cag gct cca gta ttc cgt gat gac acg tgt gtt ttc gtt 1296

Leu Asp Arg Gin Ala Pro Val Phe Arg Asp Asp Thr Cys Val Phe Val

420 425 430 tct cag tct ggc gag acc gct gac tcc ctt atg gcc ttg cgc tac tgt 1344

Ser Gin Ser Gly Glu Thr Ala Asp Ser Leu Met Ala Leu Arg Tyr Cys

435 440 445 ctc gaa cgc gga gca ttg acc gtt ggt att gtt aat gtt gta ggg tcc 1392

Leu Glu Arg Gly Ala Leu Thr Val Gly Ile Val Asn Val Val Gly Ser

450 455 460 tct atc tca ctt ctc act cac tgc ggt gtg cac atc aac gct gga ccc 1440

Ser Ile Ser Leu Leu Thr His Cys Gly Val His Ile Asn Ala Gly Pro

465 470 475 480 gaa att ggg gta gct tct acc aag gcc tac acg tcc cag ttc gtt gct 1488

Glu Ile Gly Val Ala Ser Thr Lys Ala Tyr Thr Ser Gin Phe Val Ala

485 490 495 atg gtc atg ttc gct ttg tct ctt agt gag gat agg gcg tct aag cag 1536

Met Val Met Phe Ala Leu Ser Leu Ser Glu Asp Arg Ala Ser Lys Gin

500 505 510 aag agg cgt gag gag att atg gag ggc ctt tct aag gtc tcc gag caa 1584

Lys Arg Arg Glu Glu Ile Met Glu Gly Leu Ser Lys Val Ser Glu Gin

515 520 525 ttc aag gag atc ttg aaa ctt aac gaa ccc atc aaa caa ttg tgt gca 1632

Phe Lys Glu Ile Leu Lys Leu Asn Glu Pro Ile Lys Gin Leu Cys Ala

530 535 540 aac ttc aag aat caa aag agt ttg ctt ctg ctg ggc agg ggt ggt caa 1680

Asn Phe Lys Asn Gin Lys Ser Leu Leu Leu Leu Gly Arg Gly Gly Gin 545 550 555 560 ttc cct act gcc ctt gaa ggt gca ctt aaa atc aaa gag atc tct tat 1728 Phe Pro Thr Ala Leu Glu Gly Ala Leu Lys Ile Lys Glu Ile Ser Tyr 565 570 575 ctc cat tgc gag gct gtc atg tec ggc gag ttg aag cat ggt gtt ctt 1776

Leu His Cys Glu Ala Val Met Ser Gly Glu Leu Lys His Gly Val Leu 580 585 590 gcc ctt gtc gat gaa aac ttg ccc atc atc atg att ctt acg agg gac 1824

Ala Leu Val Asp Glu Asn Leu Pro Ile Ile Met Ile Leu Thr Arg Asp 595 600 605 aac ctg ttc acg aag tcg ttg aat gct tat caa caa gtc att gcc aga 1872

Asn Leu Phe Thr Lys Ser Leu Asn Ala Tyr Gin Gin Val Ile Ala Arg 610 615 620 ggt ggc cgt cct att gta att tgt aat tec gat gac ccg gaa ttt tct 1920 Gly Gly Arg Pro Ile Val Ile Cys Asn Ser Asp Asp Pro Glu Phe Ser 625 630 635 640 tcg gct caa acc gtg aag att gaa gtt cca aag act gtc gat tgc ctg 1968 Ser Ala Gin Thr Val Lys Ile Glu Val Pro Lys Thr Val Asp Cys Leu 645 650 655 caa ggt ctc ctg aat gtc atc ccc ttg cag tta atc gct tac tgg ttg 2016

Gin Gly Leu Leu Asn Val Ile Pro Leu Gin Leu Ile Ala Tyr Trp Leu 660 665 670 gct gtg tct gag ggt ctc aat gtt gat ttc cct cgc aat ctt gcg aag 2064

Ala Val Ser Glu Gly Leu Asn Val Asp Phe Pro Arg Asn Leu Ala Lys 675 680 685 tca gtc aca gtt gag taa 2082

Ser Val Thr Val Glu 690

<210> 243

<211> 693

<212> PRT

<213> Aspergillus oryzae

<400> 243

Met Cys Gly Ile Phe Gly Tyr Ile Asn Tyr Leu Val Glu Arg Asp Arg

1 5 10 15

Lys Phe Ile Leu Asp Thr Leu Leu Asn Gly Leu Ser Arg Leu Glu Tyr 20 25 30

Arg Gly Tyr Asp Ser Ala Gly Leu Ala Ile Asp Gly Asp Lys Lys Asn 35 40 45

Glu Val Cys Ala Phe Lys Glu Val Gly Lys Val Ala Lys Leu Arg Glu 50 55 60

Leu Ile Asp Glu Cys Lys Pro Asp Leu Thr Lys Ser Phe Glu Ser His 65 70 75 80

Ala Gly Ile Ser His Thr Arg Trp Ala Thr His Gly Thr Pro Ser Arg 85 90 95

Leu Asn Cys His Pro His Arg Ser Asp Leu Asn Trp Glu Phe Ser Val 100 105 110 Val His Asn Gly Ile Ile Thr Asn Tyr Lys Glu Leu Lys Ala Leu Leu 115 120 125

Glu Ser Lys Gly Phe Arg Phe Glu Thr Asp Thr Asp Thr Glu Cys Ile

130 135 140

Ala Lys Leu Thr Lys Tyr Leu Tyr Asp Gin Gin Pro Asp Ile Glu Phe 145 150 155 160

Thr Val Leu Ala Lys Ala Val Val Lys Glu Leu Glu Gly Ala Phe Gly

165 170 175

Leu Leu Ile Lys Ser Val His Tyr Pro His Glu Val Ile Ala Ala Arg 180 185 190

Lys Gly Ser Pro Leu Val Ile Gly Val Arg Thr Ser Lys Lys Met Lys 195 200 205 Val Asp Phe Val Asp Val Glu Tyr Ser Glu Asp Gly Ala Leu Pro Ala

210 215 220

Glu Gin Ala Ser Gin Asn Val Ala Ile Lys Lys Ser Ala Thr Gly Leu 225 230 235 240

Leu Ala Pro Pro Asp Lys Ser Leu Leu His Arg Ser Gin Ser Arg Ala

245 250 255

Phe Leu Ser Asp Asp Gly Ile Pro Gin Pro Ala Glu Phe Phe Leu Ser 260 265 270

Ser Asp Pro Ser Ala Ile Val Glu His Thr Lys Lys Val Leu Tyr Leu 275 280 285

Glu Asp Asp Asp Ile Ala His Val His Glu Gly Gin Leu Asn Ile His

290 295 300

Arg Leu Thr Lys Asp Asp Gly Thr Ser Asn Val Arg Ala Ile Gin Thr 305 310 315 320 Ile Glu Leu Glu Leu Gin Glu Ile Met Lys Gly Lys Phe Asp His Phe

325 330 335

Met Gin Lys Glu Ile Phe Glu Gin Pro Glu Ser Val Ile Asn Thr Met 340 345 350

Arg Gly Arg Leu Asp Val Ala Asn Lys Gin Val Thr Leu Gly Gly Leu 355 360 365

Arg Gin Tyr Ile Ser Thr Ile Arg Arg Cys Arg Arg Ile Ile Phe Val

370 375 380

Ala Cys Gly Thr Ser Tyr His Ser Cys Met Ala Val Arg Gly Val Phe 385 390 395 400

Glu Glu Leu Thr Glu Ile Pro Ile Ser Val Glu Leu Ala Ser Asp Phe

405 410 415

Leu Asp Arg Gin Ala Pro Val Phe Arg Asp Asp Thr Cys Val Phe Val 420 425 430

Ser Gin Ser Gly Glu Thr Ala Asp Ser Leu Met Ala Leu Arg Tyr Cys

435 440 445

Leu Glu Arg Gly Ala Leu Thr Val Gly Ile Val Asn Val Val Gly Ser

450 455 460

Ser Ile Ser Leu Leu Thr His Cys Gly Val His Ile Asn Ala Gly Pro 465 470 475 480

Glu Ile Gly Val Ala Ser Thr Lys Ala Tyr Thr Ser Gin Phe Val Ala

485 490 495

Met Val Met Phe Ala Leu Ser Leu Ser Glu Asp Arg Ala Ser Lys Gin 500 505 510

Lys Arg Arg Glu Glu Ile Met Glu Gly Leu Ser Lys Val Ser Glu Gin 515 520 525

Phe Lys Glu Ile Leu Lys Leu Asn Glu Pro Ile Lys Gin Leu Cys Ala

530 535 540

Asn Phe Lys Asn Gin Lys Ser Leu Leu Leu Leu Gly Arg Gly Gly Gin 545 550 555 560

Phe Pro Thr Ala Leu Glu Gly Ala Leu Lys Ile Lys Glu Ile Ser Tyr

565 570 575 Leu His Cys Glu Ala Val Met Ser Gly Glu Leu Lys His Gly Val Leu 580 585 590

Ala Leu Val Asp Glu Asn Leu Pro Ile Ile Met Ile Leu Thr Arg Asp 595 600 605

Asn Leu Phe Thr Lys Ser Leu Asn Ala Tyr Gin Gin Val Ile Ala Arg

610 615 620

Gly Gly Arg Pro Ile Val Ile Cys Asn Ser Asp Asp Pro Glu Phe Ser 625 630 635 640

Ser Ala Gin Thr Val Lys Ile Glu Val Pro Lys Thr Val Asp Cys Leu

645 650 655

Gin Gly Leu Leu Asn Val Ile Pro Leu Gin Leu Ile Ala Tyr Trp Leu 660 665 670

Ala Val Ser Glu Gly Leu Asn Val Asp Phe Pro Arg Asn Leu Ala Lys 675 680 685

Ser Val Thr Val Glu

690

<210> 244

<211> 1347

<212> DNA

<213> Aspergillus oryzae

<220>

<221> CDS

<222> (1) . . (1347 )

<223> TUB1 Aspergillus essential gene (mRNA)

<400> 244 atg aga gaa gtt att agt ttg aac gtt ggt cag gct ggt tgc cag atc 48

Met Arg Glu Val Ile Ser Leu Asn Val Gly Gin Ala Gly Cys Gin Ile

1 5 10 15 gcc aat tct tgc tgg gag ctc tac tgt ctt gag cac ggc atc cag ccc 96

Ala Asn Ser Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile Gin Pro

20 25 30 gat ggt tac ttg acc gag gaa cgc aag aag gaa gac cct gac cat ggt 144 Asp Gly Tyr Leu Thr Glu Glu Arg Lys Lys Glu Asp Pro Asp His Gly 35 40 45 ttc agc acc ttc ttc tcc gaa act ggc cag ggc aag tat gtt cct cgt 192

Phe Ser Thr Phe Phe Ser Glu Thr Gly Gin Gly Lys Tyr Val Pro Arg 50 55 60 acc atc tac gcc gat ctg gag ccc aat gtt gtc gat gag gtc cgc act 240 Thr Ile Tyr Ala Asp Leu Glu Pro Asn Val Val Asp Glu Val Arg Thr 65 70 75 80 ggc acc tac cgt acc ctt ttc cac ccc gag aac atg atc acc ggc aag 288

Gly Thr Tyr Arg Thr Leu Phe His Pro Glu Asn Met Ile Thr Gly Lys

85 90 95 gag gat gcc tcg aac aac tat gcc cgt ggt cac tac acc gtt ggc aag 336

Glu Asp Ala Ser Asn Asn Tyr Ala Arg Gly His Tyr Thr Val Gly Lys

100 105 110 gag atg atc gac cag gtc ctc gac aag gtt cgc cgt gtg gcc gac aac 384

Glu Met Ile Asp Gin Val Leu Asp Lys Val Arg Arg Val Ala Asp Asn

115 120 125 tgc gct ggt ctc cag ggc ttc ctc gtc ttc cac tct ttc ggt ggt ggt 432

Cys Ala Gly Leu Gin Gly Phe Leu Val Phe His Ser Phe Gly Gly Gly

130 135 140 act ggt tcc ggt ttc ggt gct ctc ctg atg gag cgt ctg tct gtg gac 480

Thr Gly Ser Gly Phe Gly Ala Leu Leu Met Glu Arg Leu Ser Val Asp

145 150 155 160 tac ggc aag aag tcc aag ctg gag ttc tgc gtc tac cct gcc ccc cag 528

Tyr Gly Lys Lys Ser Lys Leu Glu Phe Cys Val Tyr Pro Ala Pro Gin

165 170 175 aat gcc acc tcc gtc gtt gag ccc tac aac tcc atc ctg act acc cac 576

Asn Ala Thr Ser Val Val Glu Pro Tyr Asn Ser Ile Leu Thr Thr His

180 185 190 acc acc ctt gag cac tcc gac tgc agt ttc atg gtt gac aac gaa gcc 624

Thr Thr Leu Glu His Ser Asp Cys Ser Phe Met Val Asp Asn Glu Ala

195 200 205 atc tac gac atc tgc cgc cgc aac ctt ggc atc gag cgt ccc agc tat 672 Ile Tyr Asp Ile Cys Arg Arg Asn Leu Gly Ile Glu Arg Pro Ser Tyr

210 215 220 gag aac ctg aac cgc ctg att gct cag gtt gtc tcc tcc atc acc gcc 720

Glu Asn Leu Asn Arg Leu Ile Ala Gin Val Val Ser Ser Ile Thr Ala

225 230 235 240 tcc ctg cgt ttc gat ggt tcc ctg aac gtg gat ctc aac gag ttc cag 768

Ser Leu Arg Phe Asp Gly Ser Leu Asn Val Asp Leu Asn Glu Phe Gin

245 250 255 acc aac ctg gtt ccc tac ccc cgt att cac ttc cct ctc gtt gcc tat 816

Thr Asn Leu Val Pro Tyr Pro Arg Ile His Phe Pro Leu Val Ala Tyr

260 265 270 gct ccc gtc atc tcc gcg gcc aag gcc tcc cac gag gcc aac tcc gtc 864

Ala Pro Val Ile Ser Ala Ala Lys Ala Ser His Glu Ala Asn Ser Val

275 280 285 aac gag atc acc atg tct tgc ttc gag ccc aac aac cag atg gtc aag 912

Asn Glu Ile Thr Met Ser Cys Phe Glu Pro Asn Asn Gin Met Val Lys

290 295 300 tgt gac ccc cgc aat ggc aag tac atg gct act tgc ttg ctg tac cgt 960

Cys Asp Pro Arg Asn Gly Lys Tyr Met Ala Thr Cys Leu Leu Tyr Arg

305 310 315 320 ggt gat gtt gtg ccc aag gag acc cac gcc gcc gtt gct acc ctc aag 1008 Gly Asp Val Val Pro Lys Glu Thr His Ala Ala Val Ala Thr Leu Lys 325 330 335 acc aag cgt acc atc cag ttc gtc gac tgg tgc cct act ggt ttc aag 1056

Thr Lys Arg Thr Ile Gin Phe Val Asp Trp Cys Pro Thr Gly Phe Lys 340 345 350 atc ggt atc tgc tac cag ccc cct aag cag gtt ccc aac ggt gac ctt 1104 Ile Gly Ile Cys Tyr Gin Pro Pro Lys Gin Val Pro Asn Gly Asp Leu 355 360 365 gcc aac ctc agc cgt gct gtc tgc atg ctg tct aac acc acc gcc atc 1152

Ala Asn Leu Ser Arg Ala Val Cys Met Leu Ser Asn Thr Thr Ala Ile 370 375 380 tcc gag gcc tgg tcc gct ctc gac cac aag ttc gat ctc atg tac tcc 1200 Ser Glu Ala Trp Ser Ala Leu Asp His Lys Phe Asp Leu Met Tyr Ser 385 390 395 400 aag cgt gct ttc gtt cac tgg tat gtt gga gag ggt atg gag gag ggt 1248 Lys Arg Ala Phe Val His Trp Tyr Val Gly Glu Gly Met Glu Glu Gly 405 410 415 gaa ttc tcc gag gcc cgt gag gac ctg gct gcc ctc gag cgc gac tac 1296

Glu Phe Ser Glu Ala Arg Glu Asp Leu Ala Ala Leu Glu Arg Asp Tyr 420 425 430 gag gag gtc gcc agc gac tcg ctg gag gag gag gtt gag gct gag tac 1344

Glu Glu Val Ala Ser Asp Ser Leu Glu Glu Glu Val Glu Ala Glu Tyr 435 440 445 taa 1347

<210> 245

<211> 448

<212> PRT

<213> Aspergillus oryzae

<400> 245

Met Arg Glu Val Ile Ser Leu Asn Val Gly Gin Ala Gly Cys Gin Ile

1 5 10 15

Ala Asn Ser Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile Gin Pro 20 25 30

Asp Gly Tyr Leu Thr Glu Glu Arg Lys Lys Glu Asp Pro Asp His Gly 35 40 45

Phe Ser Thr Phe Phe Ser Glu Thr Gly Gin Gly Lys Tyr Val Pro Arg

50 55 60

Thr Ile Tyr Ala Asp Leu Glu Pro Asn Val Val Asp Glu Val Arg Thr 65 70 75 80

Gly Thr Tyr Arg Thr Leu Phe His Pro Glu Asn Met Ile Thr Gly Lys 85 90 95 Glu Asp Ala Ser Asn Asn Tyr Ala Arg Gly His Tyr Thr Val Gly Lys

100 105 110

Glu Met Ile Asp Gin Val Leu Asp Lys Val Arg Arg Val Ala Asp Asn 115 120 125

Cys Ala Gly Leu Gin Gly Phe Leu Val Phe His Ser Phe Gly Gly Gly

130 135 140

Thr Gly Ser Gly Phe Gly Ala Leu Leu Met Glu Arg Leu Ser Val Asp 145 150 155 160

Tyr Gly Lys Lys Ser Lys Leu Glu Phe Cys Val Tyr Pro Ala Pro Gin 165 170 175

Asn Ala Thr Ser Val Val Glu Pro Tyr Asn Ser Ile Leu Thr Thr His

180 185 190

Thr Thr Leu Glu His Ser Asp Cys Ser Phe Met Val Asp Asn Glu Ala 195 200 205 Ile Tyr Asp Ile Cys Arg Arg Asn Leu Gly Ile Glu Arg Pro Ser Tyr

210 215 220

Glu Asn Leu Asn Arg Leu Ile Ala Gin Val Val Ser Ser Ile Thr Ala 225 230 235 240

Ser Leu Arg Phe Asp Gly Ser Leu Asn Val Asp Leu Asn Glu Phe Gin 245 250 255

Thr Asn Leu Val Pro Tyr Pro Arg Ile His Phe Pro Leu Val Ala Tyr

260 265 270

Ala Pro Val Ile Ser Ala Ala Lys Ala Ser His Glu Ala Asn Ser Val 275 280 285

Asn Glu Ile Thr Met Ser Cys Phe Glu Pro Asn Asn Gin Met Val Lys

290 295 300

Cys Asp Pro Arg Asn Gly Lys Tyr Met Ala Thr Cys Leu Leu Tyr Arg 305 310 315 320

Gly Asp Val Val Pro Lys Glu Thr His Ala Ala Val Ala Thr Leu Lys 325 330 335

Thr Lys Arg Thr Ile Gin Phe Val Asp Trp Cys Pro Thr Gly Phe Lys 340 345 350 Ile Gly Ile Cys Tyr Gin Pro Pro Lys Gin Val Pro Asn Gly Asp Leu

355 360 365

Ala Asn Leu Ser Arg Ala Val Cys Met Leu Ser Asn Thr Thr Ala Ile

370 375 380

Ser Glu Ala Trp Ser Ala Leu Asp His Lys Phe Asp Leu Met Tyr Ser 385 390 395 400

Lys Arg Ala Phe Val His Trp Tyr Val Gly Glu Gly Met Glu Glu Gly

405 410 415

Glu Phe Ser Glu Ala Arg Glu Asp Leu Ala Ala Leu Glu Arg Asp Tyr 420 425 430

Glu Glu Val Ala Ser Asp Ser Leu Glu Glu Glu Val Glu Ala Glu Tyr

435 440 445

<210> 246

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> agsA

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5474 )

<223> essential gene ERG10

<220>

<221> exon

<222> (4010) . . (4074 )

<220>

<221> exon

<222> (4142 ) . . (4191)

<220>

<221> exon <222> (4249) . . (4324 )

<220>

<221> exon

<222> (4398) . . (4887 )

<220>

<221> exon

<222> (4956) . . (5474 )

<400> 246 aacgcgacgt tgcccttttc tgggcttagt cgccattttt cgccttgtcc aggcaagaca 60 ctttcaatga cagtgttgac ttcttcaatc acatgcttga gcttgaaata ggcgtcgttg 120 ggtggtatct tcagttcgag gatcagccga tccattttgt tcacaaccat ggtcaacgga 180 aggccctcta gcacagcgtg cttgatgatt tgctccgtat tcgattgtac tccttccaca 240 atatcaacga ctatcaccac gccatcaacc agtcgtgatg aggcagctac ttcatcaaca 300 aaattcacat gccccggggt atcaagaatg ttgaacaaat gagacttccc cttagtgccc 360 tgaagaacta ggctcatagg agctgattta atagaaaggc ctcgctctct ctctagaaag 420 tgaatatcgg tataacgaag ctgtacttcc tttcttttac ctgctcgctt ctctagccgt 480 tcagtcaaat catgcgtttg cataacaagc atatccatga acgcggtctt cccatgatgt 540 aaatgaccga caagcgcaat gttccttatt tgttcaggaa aattcagaag atcagtcatg 600 aattcccttg aaaagtgaac ccgggggagc tcagtctctt caatggcgaa cttcttttgc 660 tgtacggggg caacgatagg ctcggagaga ggctgtgcgt cttcttcttg aactaatgtc 720 tctacctctg tcccgtagac ctgttgtgcg ctagggtagt attgtttgtc ttcgtgtaga 780 atgacggcat ttgagggggc ttctgtaatg agatatatca gcaccgacta aatccagggg 840 ctttaagaga gtgcccaccg tccacctcca tagactgctg cccatataat tcttcatctt 900 cttcctcccc aaacgcctcg ttgaaggcga aagcccgagg cttcacttcg tcgtgttggc 960 cgtcttcatc tgaatctgca gcctctccaa tgtagttgcc aaacctgtgt ggtcttgatt 1020 agaatttgca caattagagc gttagattgg atgcaactta ctcatcgtag agatcatcca 1080 tcgtgcggtg ggaaaggtgg ttcaagctgc caaaaacttg tcaccagtga gctgtcgcat 1140 ctctttttgt cccaagctgc tgaagtccgt ctaatggact caccgagtgg tccgtgcgtc 1200 gaccctacca ttttggtagc cccgccgcgc ggatgccacg tccaggaggg gtccttaaat 1260 gactttgatt tctcaccacc tcttctcgtg tcagagatac ctcactgcta tcgtcggctg 1320 aataactcaa ctctttcttg tttcccttct tccccccttt aatcccacca atcttcttta 1380 ttaccggcaa gttctgtcta cacgggtgcc ctttaccaca caccgaaccc ctttaaacgc 1440 ttcacgctac agaagctttt ggtttacgac tcttctaatc agctatcgta ggccgccgat 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggtg ttgtttctta atataggcac acggatcccg ccatcgatcg 3300 gcgccctgtt cgagtatggc catcctcaca cattgtcggg ctccgctggc gctgcctcaa 3360 catattttta aatggggaaa ccataagcaa agagactcca tcgggcgcac tacacatctc 3420 gcctcttctg gaccctgtgg atcgggactg gggtggatgg caaaccgtca agttgccgcc 3480 gcttcttcgc gctgttgcgg cgtcacgcca tataggcgac tctcccaatc atggaaagce 3540 tgcgttccag cgatattcaa cgacgtggtg gcttggattg cagcttcgac cagatcacag 3600 caatagaata gcgatgatte cagccgtggg cctttagggt agaaaccata ctgggctagg 3660 aaacatgccg tatcaagtga gaaacaccgc taccccacca eggctgaaaa gtagatagtg 3720 gatggttcca tgtcatctat cagcaggcga ggagtgggtc tatagtagag gttgtaatgg 3780 tcgtggtaga ggtccctggc ccatttttat ccccatcctt cccggcctga tggttccaag 3840 ccttctttac gaaaagtttt gcagctgctt tttcgagtcg acaggtcgct cgttccttcg 3900 tttgatttcc ctcgcttttg accacgccca gggcatgtag gatttattcc gtttgagatt 3960 atcgagagce taagtcgcge gcagttgtcg gtagcaggcc aetgccggg atg tct tct 4018 Met Ser Ser 1 ctt ccg ccg gtc tat att gtt tcc tct gcc cgc acc cca gtc ggc tct 4066 Leu Pro Pro Val Tyr Ile Val Ser Ser Ala Arg Thr Pro Val Gly Ser 5 10 15 ttc ttg gg gtgagattgg ccttgtgcct tgtctatacg tacaatattg 4114 Phe Leu Gly 20 acttacactg actatccgct tccacag g tcg ctc tca agt ctc act gcc ccg 4166 Ser Leu Ser Ser Leu Thr Ala Pro 25 30 cag tta ggc tct cat gct att aaa g gtatgacagc tctcgagtgg 4211 Gin Leu Gly Ser His Ala Ile Lys

35 eggtaegtgt tgatatacta acaaactcga aattcag ct gcg ctc agc aaa gcg 4265 Ala Ala Leu Ser Lys Ala 40 gat gga atc aag ccg tct gat atc cag gag gtc ttc ttt ggc aat gtc 4313 Asp Gly Ile Lys Pro Ser Asp Ile Gin Glu Val Phe Phe Gly Asn Val 45 50 55 60 atc tcc gca aa gtgagtcaac gtccaccctt ataccttata gccaacatcg 4364 Ile Ser Ala Asn aaggcacact tacattctcg ttctatggat cag c gtt gga caa aat cct gct 4416 Val Gly Gin Asn Pro Ala 65 70 aga cag tgt gct ctc ggc gct ggt ctc aat gaa tca act gtc tgt act 4464 Arg Gin Cys Ala Leu Gly Ala Gly Leu Asn Glu Ser Thr Val Cys Thr 75 80 85 acg gtt aat aag gtg tgc gcg tct ggc ttg aaa gcg gtt att ctc ggt 4512 Thr Val Asn Lys Val Cys Ala Ser Gly Leu Lys Ala Val Ile Leu Gly 90 95 100 gca cag acc atc atg act ggc aat gcg gat att gtc gta gca ggc ggt 4560 Ala Gin Thr Ile Met Thr Gly Asn Ala Asp Ile Val Val Ala Gly Gly 105 110 115 gct gaa tcc atg tct aac gcc cct cat tac ctt cca aac ctt cgc gtc 4608

Ala Glu Ser Met Ser Asn Ala Pro His Tyr Leu Pro Asn Leu Arg Val 120 125 130 ggt gcg aaa tac ggc aac cag agt ctg gtg gac ggt att atg aag gat 4656

Gly Ala Lys Tyr Gly Asn Gin Ser Leu Val Asp Gly Ile Met Lys Asp

135 140 145 150 ggc ttg aca gac gca gga aag cag gaa ctc atg ggc ttg caa gcc gag 4704

Gly Leu Thr Asp Ala Gly Lys Gin Glu Leu Met Gly Leu Gin Ala Glu

155 160 165 gag tgt gct cag gat cat ggc ttt agc agg gaa caa cag gat gat tat 4752

Glu Cys Ala Gin Asp His Gly Phe Ser Arg Glu Gin Gin Asp Asp Tyr

170 175 180 gcc att cgc act tac gaa aaa gca cag gcg gct caa aag gct ggc ctt 4800

Ala Ile Arg Thr Tyr Glu Lys Ala Gin Ala Ala Gin Lys Ala Gly Leu

185 190 195 ttt gac gaa gaa att gcg cct att gaa ctt cct ggc ttt agg ggc aag 4848

Phe Asp Glu Glu Ile Ala Pro Ile Glu Leu Pro Gly Phe Arg Gly Lys

200 205 210 cca ggt gtg act gtg tca caa gac gaa gaa cca aag aat gtgaggaaga 4897

Pro Gly Val Thr Val Ser Gin Asp Glu Glu Pro Lys Asn

215 220 225 tattacttte tacagatttt ttaagtcgte ggccactgct aatatactca ttgcgtag 4955 ctt aac ccg gat aag ctt cga gct atc aag cct gca ttt atc ccc gga 5003

Leu Asn Pro Asp Lys Leu Arg Ala Ile Lys Pro Ala Phe Ile Pro Gly

230 235 240 tcc ggc acg gtc aca gcc ccg aat tcc tca cct ctt aac gac ggt gct 5051

Ser Gly Thr Val Thr Ala Pro Asn Ser Ser Pro Leu Asn Asp Gly Ala

245 250 255 gct gct gtt atc ctc gtc tca gaa gct aaa ctg aaa gag ctt aac cta 5099

Ala Ala Val Ile Leu Val Ser Glu Ala Lys Leu Lys Glu Leu Asn Leu

260 265 270 275 aag cct gtt gca aag att ctt ggc tgg gga gat gcc gcc cag cag cca 5147

Lys Pro Val Ala Lys Ile Leu Gly Trp Gly Asp Ala Ala Gin Gin Pro

280 285 290 agc aaa ttc aca act gcc cca gct cta gca att ccc aag gcc ctc agc 5195

Ser Lys Phe Thr Thr Ala Pro Ala Leu Ala Ile Pro Lys Ala Leu Ser

295 300 305 cat gca ggt gtg gct cag gat gct gtt gat gcg ttc gag att aac gaa 5243

His Ala Gly Val Ala Gin Asp Ala Val Asp Ala Phe Glu Ile Asn Glu

310 315 320 gcg ttc agc gta gtt gct ctg gcc aat atg aaa ctc ctg ggg ttg gct 5291

Ala Phe Ser Val Val Ala Leu Ala Asn Met Lys Leu Leu Gly Leu Ala

325 330 335 gaa gat aaa gtc aac atc cat ggt ggt gca gtg gct atc ggt cat cct 5339

Glu Asp Lys Val Asn Ile His Gly Gly Ala Val Ala Ile Gly His Pro

340 345 350 355 atc ggc gcc agc ggt gct cgt atc ttg act aca ttg ctc ggt gta ttg 5387 Ile Gly Ala Ser Gly Ala Arg Ile Leu Thr Thr Leu Leu Gly Val Leu 360 365 370 aaa gcg aga aag ggt aag att ggt tgt gcc ggg att tgt aat gga gga 5435 Lys Ala Arg Lys Gly Lys Ile Gly Cys Ala Gly Ile Cys Asn Gly Gly 375 380 385 ggt ggt gct agc gct att gtt gtc gaa tct ctc gtc tga gtcatgecga 5484 Gly Gly Ala Ser Ala Ile Val Val Glu Ser Leu Val 390 395 gtcgcccttg atcttggtat ggaat 5509

<210> 247

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> agsA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5509)

<223> essential gene FKS2

<220>

<221> exon

<222> (4010) . . (4014 )

<220>

<221> exon

<222> (4072 ) . . (4142 )

<220>

<221> exon

<222> (4229) . . (4460)

<220>

<221> exon

<222> (4534 ) . . (5509)

<400> 247 ataaaaattt cgccaccgaa gaagtatcaa ggaaactgtc tagcgagagg gactggactg 60 gaacacttga gtattcggta tccagttaag gtgatttaag ccaccccttc tcccttcaat 120 ccctaccccc cttcaaaaag gggaagaaca gtagctaaag gtagttgcca tcaagaactc 180 atacttaaaa acacgaggta agaggcccgt aatagtaatt ggttgcaaga gaaaaggggg 240 gttatccttc tgagcatcac agttttgatt acgggaagaa ccagaatatg aacgtggcaa 300 cagcggctgt cggtaccaga ccaggccttt attggatacc ggaggagtcc ctctttcgtg 360 tgtttcctac cccttttaca gtacaattac aacctacata atgtcactga tagatattta 420 tacgtgtgcc agaagatact tatgggttag tcaatgtaaa tagcttctaa ttttgattca 480 tgattgaggg gtctaaaatt ggactgaacc taatcctact caagtaggag ttagatgacg 540 atggtgagtg tgaaatgcca ggattaaggg tagcatatca gccgcacagg aactaccgag 600 ggggtggagg ctaacagccc tctttttttc tttccctttt tcctcatttt cctctctctt 660 ctcccgatga tgacgatttg tagattcata agttatagaa aaatatttgt tctatgcaca 720 cgctaatagg acatagtact gcttattgta tgtatatgcc tggtgtttaa agaagtagac 780 ttcttgagtt ccgtacggag cactatttta caatacgaat aaaaaaaatc catggagcag 840 ggaagggaag gatatagcta gctaggtccc taatagtcga tatgttattt ttgatctgtt 900 agtggagcca ctgacctgtt gaatttttgg taggtaaact tagcaacatg tccatgccat 960 aaaaggtctc caaccgagag tcacgagtga aacataccag taggtccaac aaactgtgta 1020 aggtgcagaa aaggcacatt attggccagc tccatgttga atgaatggct gcgccctgga 1080 gccggtccat agtagcttcc ctggtctctg tgtgggccga tctcacaaca taaaacatag 1140 ctcttccccc tccttccaac agattgtcga tctactcaat ttcccctttg ctatcaactt 1200 ctccccctcc tcgcttcctt ctctcactcg ccacaccgcc ctttaagaat atcacaagta 1260 atattctagc tggattatcc aaagtgtgtt ttatgttgtc cttccatggt atcttccaat 1320 tcgtttgttg acagtatatt cccttccaga gtgcactttg aagaaacccc actaccgttt 1380 acgtgacttg ctttcaatat ccggaggcaa catttgttat cgaagcatta caacgcctag 1440 tcatctcatt ctacaccttt ccacattttg actcgctctc tctcttgaca taccttaacc 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggtg ttgtttctta atataggcac acggatcccg ccatcgatcg 3300 gcgccctgtt cgagtatggc catcctcaca cattgtcggg ctccgctggc gctgcctcaa 3360 catattttta aatggggaaa ccataagcaa agagactcca tcgggcgcac tacacatctc 3420 gcctcttctg gaccctgtgg atcgggactg gggtggatgg caaaccgtca agttgccgcc 3480 gcttcttcgc gctgttgcgg cgtcacgcca tataggcgac tctcccaatc atggaaagcc 3540 tgcgttccag cgatattcaa cgacgtggtg gcttggattg cagcttcgac cagatcacag 3600 caatagaata gcgatgattc cagccgtggg cctttagggt agaaaccata ctgggctagg 3660 aaacatgccg tatcaagtga gaaacaccgc taccccacca cggctgaaaa gtagatagtg 3720 gatggttcca tgtcatctat cagcaggcga ggagtgggtc tatagtagag gttgtaatgg 3780 tcgtggtaga ggtccctggc ccatttttat ccccatcctt cccggcctga tggttccaag 3840 ccttctttac gaaaagtttt gcagctgctt tttcgagtcg acaggtcgct cgttccttcg 3900 tttgatttcc ctcgcttttg accacgccca gggcatgtag gatttattcc gtttgagatt 3960 atcgagagcc taagtcgcgc gcagttgtcg gtagcaggcc actgccggg atg tg 4014

Met Cys 1 gtacgttccc gaagacccag gcttactgaa tgtttgatgt aactaattga cccctag c 4072 ggc atc ttc ggc tac att aac tac ctc gtc gag agg gac cgc aag ttt 4120

Gly Ile Phe Gly Tyr Ile Asn Tyr Leu Val Glu Arg Asp Arg Lys Phe

5 10 15 att ctt gac act ctt ctc aat g gttggcattc ctgttgtttt caccctcact 4172 Ile Leu Asp Thr Leu Leu Asn 20 25 ggaccttgtt tactctggtt ttgggagtgt ttgtgtattc ctctgacata ttgaag ga 4230 Gly ctc tcc cgt ctt gaa tac cga ggc tac gac tct gcg ggt ctt gcc ata 4278

Leu Ser Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala Gly Leu Ala Ile

30 35 40 gat ggc gat aag aag aat gaa gtt tgt gct ttc aag gaa gtg ggc aag 4326

Asp Gly Asp Lys Lys Asn Glu Val Cys Ala Phe Lys Glu Val Gly Lys

45 50 55 gtt gct aag ctt cga gag ctt att gat gag tgc aag cct gac ttg acc 4374 Val Ala Lys Leu Arg Glu Leu Ile Asp Glu Cys Lys Pro Asp Leu Thr

60 65 70 aaa tcc ttt gaa tct cat gct ggt att tct cac act cgt tgg gcc act 4422

Lys Ser Phe Glu Ser His Ala Gly Ile Ser His Thr Arg Trp Ala Thr

75 80 85 90 cat ggg acg cct tcc cgc ttg aac tgc cac ccc cac ag gtgagtecga 4470

His Gly Thr Pro Ser Arg Leu Asn Cys His Pro His Arg

95 100 tgttccacgg tttaatcagc tcgactagga tcaggcttgt ttgctaattt tcctggcacg 4530 cag g tcc gat ctg aac tgg gaa ttt tca gtt gtc cac aat ggt atc att 4579

Ser Asp Leu Asn Trp Glu Phe Ser Val Val His Asn Gly Ile Ile 105 110 115 acc aac tac aaa gag ctc aag gct ttg ttg gag agc aag gga ttc cgc 4627

Thr Asn Tyr Lys Glu Leu Lys Ala Leu Leu Glu Ser Lys Gly Phe Arg

120 125 130 ttt gag act gac aca gac acc gaa tgt att gca aag ctt acc aaa tat 4675

Phe Glu Thr Asp Thr Asp Thr Glu Cys Ile Ala Lys Leu Thr Lys Tyr

135 140 145 150 ctc tat gac caa cag ccc gat atc gaa ttc aca gtt ttg gct aag gct 4723

Leu Tyr Asp Gin Gin Pro Asp Ile Glu Phe Thr Val Leu Ala Lys Ala

155 160 165 gtt gtg aag gag ctt gag ggc gcg ttt ggg ctt ctc atc aaa tcc gta 4771 Val Val Lys Glu Leu Glu Gly Ala Phe Gly Leu Leu Ile Lys Ser Val

170 175 180 cat tat ccc cat gag gtc att gcg gct cgc aag ggg tcg ccg ctt gtt 4819 His Tyr Pro His Glu Val Ile Ala Ala Arg Lys Gly Ser Pro Leu Val 185 190 195 att ggc gtg aga acg tcg aag aag atg aag gtg gat ttc gtg gac gtc 4867 Ile Gly Val Arg Thr Ser Lys Lys Met Lys Val Asp Phe Val Asp Val

200 205 210 gaa tac tca gaa gat gga gcc ctt cct gca gag cag gcc tec cag aac 4915

Glu Tyr Ser Glu Asp Gly Ala Leu Pro Ala Glu Gin Ala Ser Gin Asn

215 220 225 230 gtg gcc atc aag aag tct gcg acc ggc ctc ctt gcc cca cct gac aaa 4963 Val Ala Ile Lys Lys Ser Ala Thr Gly Leu Leu Ala Pro Pro Asp Lys

235 240 245 tcc ctt ttg cat agg tca cag tct cgc gct ttc ctt tec gat gat ggc 5011

Ser Leu Leu His Arg Ser Gin Ser Arg Ala Phe Leu Ser Asp Asp Gly

250 255 260 atc ccc cag cca gct gag ttt ttc ttg tct tct gac cca tct gca att 5059 Ile Pro Gin Pro Ala Glu Phe Phe Leu Ser Ser Asp Pro Ser Ala Ile

265 270 275 gtc gag cat acg aag aag gtc ctc tat ctc gaa gat gat gat atc gct 5107 Val Glu His Thr Lys Lys Val Leu Tyr Leu Glu Asp Asp Asp Ile Ala

280 285 290 cat gtg cat gag gga cag tta aac att cat cgc ctc aca aaa gat gat 5155

His Val His Glu Gly Gin Leu Asn Ile His Arg Leu Thr Lys Asp Asp

295 300 305 310 ggt act tct aat gtt cgc gct att cag aca att gaa ctc gaa ttg cag 5203

Gly Thr Ser Asn Val Arg Ala Ile Gin Thr Ile Glu Leu Glu Leu Gin

315 320 325 gag att atg aag ggc aag ttt gac cac ttc atg caa aag gaa att ttc 5251

Glu Ile Met Lys Gly Lys Phe Asp His Phe Met Gin Lys Glu Ile Phe

330 335 340 gag caa ccc gag tct gtg atc aat acc atg aga gga cga ctg gat gtt 5299

Glu Gin Pro Glu Ser Val Ile Asn Thr Met Arg Gly Arg Leu Asp Val

345 350 355 gca aac aag caa gtc aca ctc ggt ggc ctg egg cag tac att tct act 5347

Ala Asn Lys Gin Val Thr Leu Gly Gly Leu Arg Gin Tyr Ile Ser Thr

360 365 370 atc cgc cgc tgc aga aga atc ata ttt gtt gcc tgt gga act agt tac 5395 Ile Arg Arg Cys Arg Arg Ile Ile Phe Val Ala Cys Gly Thr Ser Tyr

375 380 385 390 cat tca tgc atg gct gtg cgt gga gtc ttt gaa gag ctt act gag att 5443

His Ser Cys Met Ala Val Arg Gly Val Phe Glu Glu Leu Thr Glu Ile

395 400 405 cct att tct gtt gaa ctt gct tec gat ttc ctg gat aga cag gct cca 5491

Pro Ile Ser Val Glu Leu Ala Ser Asp Phe Leu Asp Arg Gin Ala Pro

410 415 420 gta ttc cgt gat gac acg 5509 Val Phe Arg Asp Asp Thr

425 <210> 248

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> agsA

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5509)

<223> TUB1 essential gene

<220>

<221> exon

<222> (4010) . . (4034 )

<220>

<221> exon

<222> (4124 ) . . (4164 )

<220>

<221> exon

<222> (4239) . . (4265)

<220>

<221> exon

<222> (4337 ) . . (4412 )

<220>

<221> exon

<222> (4473) . . (4527 )

<220>

<221> exon

<222> (5481) . . (5509)

<400> 248 cgggaagatg ttccatagaa agggatcaag gatgatatcc gctgcaaccg ggggaacatt 60 ctccattagt gctttgttaa gagaaaagta gtccgccgcc gcagtaacac gaaggaaaag 120 caccgaatca gacgtaggtg cttgatcatg aaaagaggaa gatttgcgag gaacagcgtt 180 agactgaggg gaggcgagat gggcttggga atagaggcta atcgaggaga agagtgcggg 240 atcatcaatg gcttgtggaa gagtatgcgt tgtcagggta aaggcggtcg gttgctttga 300 ctacttgtca gctaattttc tttggtcgcc ttgctgggca ctggtagagg gtatatgagg 360 acgtacagtt gccaaccaac acacccgcac ctcataccgc tggccggggg tcaagttctc 420 cagaaaatac catgagtctg taccagggaa ctcgttggtc ggaaatgtag cgttaattct 480 ggttcgaaga acgggactag aggaggagag gcgatctaga cctaggtcat cgagggtagg 540 atcgaccgtg gggatgatga ggggttgcgg agcgatgaag atcgttttct cgacattggc 600 gatagcgggg aatgcgaggg tgcagagtaa gagtagatat tggagtagca tcatgacaga 660 gagttcgcta tgttcaattg acgctgctgc tagacggtca tggttctagt ccacacgcag 720 ttgagtatgg taggctatat gtatgtatgt agtaaatgag gtagatctga tactaactgt 780 tagattccta agtgaagtcc ttacatgaaa ttgattcaat gtcaatacga tcttaccata 840 aattaggcca ttgtgcgcag caatgttggt gactagaaat agagcactgt gtgtccgaga 900 cttgttctat gtgagtattt tcaatagtac ttactatcgc aagctgtacg gatagtcaaa 960 tacaatgcag ctgcacgaag agatgccgtc attgacacgt gatcgaccct tgccgtgaac 1020 taagcccaaa taagtggctg agtcagcggt gggctggaag ggcggtcacg gcatcggaca 1080 agccaatcag aaatgggatt gtgggcgacg cgacccaacg gatggatcaa caatgaaaag 1140 gtggaagtcc tgcttttggg ttgacagcgc atcgcaagac cacaacaaat tgaccgatca 1200 ttcgggtctt gggcaattgt ttaattgatc cccttagtct atttttctcc tcgtcaatca 1260 ccccaagggt ttgttctcct cccccatttc ctcccaccgg ttcccctttc cctttctttt 1320 ggttcttttc ccaggtggtg tttttttcgt ttacttaccc ctgaaacttc cttacagatc 1380 agggcatttt tccccctcta aagtccttcc acattccacc cactccgttt cacacacccc 1440 cctccttcca atcttaattc ctttgtcact ccgacattcg tcccttttag actcgacaag 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggtg ttgtttctta atataggcac acggatcccg ccatcgatcg 3300 gcgccctgtt cgagtatggc catcctcaca cattgtcggg ctccgctggc gctgcctcaa 3360 catattttta aatggggaaa ccataagcaa agagactcca tcgggcgcac tacacatctc 3420 gcctcttctg gaccctgtgg atcgggactg gggtggatgg caaaccgtca agttgccgcc 3480 gcttcttcgc gctgttgcgg cgtcacgcca tataggcgac tctcccaatc atggaaagcc 3540 tgcgttccag cgatattcaa cgacgtggtg gcttggattg cagcttcgac cagatcacag 3600 caatagaata gcgatgattc cagccgtggg cctttagggt agaaaccata ctgggctagg 3660 aaacatgccg tatcaagtga gaaacaccgc taccccacca cggctgaaaa gtagatagtg 3720 gatggttcca tgtcatctat cagcaggcga ggagtgggtc tatagtagag gttgtaatgg 3780 tcgtggtaga ggtccctggc ccatttttat ccccatcctt cccggcctga tggttccaag 3840 ccttctttac gaaaagtttt gcagctgctt tttcgagtcg acaggtcgct cgttccttcg 3900 tttgatttcc ctcgcttttg accacgccca gggcatgtag gatttattcc gtttgagatt 3960 atcgagagcc taagtcgcgc gcagttgtcg gtagcaggcc actgccggg atg aga gaa 4018 Met Arg Glu 1 gtt att agt ttg aac g gtatgtgttt tctcaatctt ttcctcacac cgcctccttc 4074 Val Ile Ser Leu Asn 5 accggagtcg gtctgttgac cgttactaat tgttttttta cgtgtgcag tt ggt cag 4131 Val Gly Gin 10 gct ggt tgc cag atc gcc aat tct tgc tgg gag gtaeggagat ggaactatac 4184 Ala Gly Cys Gin Ile Ala Asn Ser Cys Trp Glu

15 20 tagaagatag aggtgtttgg aatgtgacta ttgaaactga cattatggtt ctag ctc 4241

Leu tac tgt ctt gag cac ggc atc cag gtacgtggcc acgatgacaa tccatccatc 4295 Tyr Cys Leu Glu His Gly Ile Gin

25 30 ttcatctcca tcgggcacac agactaaccg gtatacgata g ccc gat ggt tac ttg 4351 Pro Asp Gly Tyr Leu 35 acc gag gaa cgc aag aag gaa gac cct gac cat ggt ttc agc acc ttc 4399 Thr Glu Glu Arg Lys Lys Glu Asp Pro Asp His Gly Phe Ser Thr Phe 40 45 50 ttc tcc gaa act g gtacgtcgta ttgacttgtt atcttgccgc cgagccctct 4452 Phe Ser Glu Thr

55 aacaggctat ggtgtcttag gc cag ggc aag tat gtt cct cgt acc atc tac 4504 Gly Gin Gly Lys Tyr Val Pro Arg Thr Ile Tyr 60 65 gcc gat ctg gag ccc aat gtt gt cgatgaggte cgcactggca cctaccgtac 4557 Ala Asp Leu Glu Pro Asn Val Val 70 75 ccttttccac cccgagaaca tgatcaccgg caaggaggat gcctcgaaca actatgcccg 4617 tggtcactac accgttggca aggagatgat cgaccaggtc ctcgacaagg ttcgccgtgt 4677 ggccgacaac tgcgctggtc tccagggctt cctcgtcttc cactctttcg gtggtggtac 4737 tggttccggt ttcggtgctc tcctgatgga gcgtctgtct gtggactacg gcaagaagtc 4797 caagctggag ttctgcgtct accctgcccc ccagaatgcc acctccgtcg ttgagcccta 4857 caactccatc ctgactaccc acaccaccct tgagcactcc gaetgeagtt tcatggttga 4917 caacgaagcc atctacgaca tctgccgccg caaccttggc atcgagcgte ccagctatga 4977 gaacctgaac egcctgattg ctcaggttgt ctcctccatc accgcctccc tgcgtttcga 5037 tggttccctg aacgtggatc tcaacgagtt ccagaccaac ctggttccct acccccgtat 5097 tcacttccct ctcgttgcct atgctcccgt catctccgcg gccaaggcct cccacgaggc 5157 caactccgtc aacgagatca ccatgtcttg cttcgagccc aacaaccaga tggtcaagtg 5217 tgacccccgc aatggcaagt acatggctac ttgcttgctg taccgtggtg atgttgtgcc 5277 caaggagacc cacgccgccg ttgctaccct caagaccaag cgtaccatcc agttcgtcga 5337 ctggtgccct actggtttca agatcggtat ctgctaccag ccccctaagc aggttcccaa 5397 cggtgacctt gccaacctca gccgtgctgt gtaagttccc tatctaccac tcgtgeaaga 5457 gataaaaagc tgaccagtta tag c tgc atg ctg tct aac acc acc gcc atc t 5509 Cys Met Leu Ser Asn Thr Thr Ala Ile 80

<210> 249

<211> 11018

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> bipA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5474 )

<223> ERG10 essential gene

<220>

<221> exon

<222> (4010) . . (4074 )

<220>

<221> exon

<222> (4142 ) . . (4191)

<220>

<221> exon

<222> (4249) . . (4324 )

<220>

<221> exon

<222> (4398) . . (4887 )

<220>

<221> exon

<222> (4956) . . (5474 )

<400> 249 aaegcgaegt tgcccttttc tgggcttagt cgccattttt cgccttgtcc aggcaagaca 60 ctttcaatga cagtgttgac ttcttcaatc acatgcttga gcttgaaata ggcgtcgttg 120 ggtggtatct tcagttcgag gatcagccga tccattttgt tcacaaccat ggtcaacgga 180 aggccctcta gcacagcgtg cttgatgatt tgctccgtat tcgattgtac tccttccaca 240 atatcaacga ctatcaccac gccatcaacc agtcgtgatg aggcagctac ttcatcaaca 300 aaattcacat gccccggggt atcaagaatg ttgaacaaat gagacttccc cttagtgccc 360 tgaagaacta ggctcatagg agctgattta atagaaaggc ctcgctctct ctctagaaag 420 tgaatatcgg tataacgaag ctgtacttcc tttcttttac ctgctcgctt ctctagccgt 480 tcagtcaaat catgcgtttg cataacaagc atatccatga acgcggtctt cccatgatgt 540 aaatgaccga caagcgcaat gttccttatt tgttcaggaa aattcagaag atcagtcatg 600 aattcccttg aaaagtgaac ccgggggagc tcagtctctt caatggcgaa cttcttttgc 660 tgtacggggg caacgatagg ctcggagaga ggctgtgcgt cttcttcttg aactaatgtc 720 tctacctctg tcccgtagac ctgttgtgcg ctagggtagt attgtttgtc ttcgtgtaga 780 atgacggcat ttgagggggc ttctgtaatg agatatatca gcaccgacta aatccagggg 840 ctttaagaga gtgcccaccg tccacctcca tagactgctg cccatataat tcttcatctt 900 cttcctcccc aaacgcctcg ttgaaggcga aagcccgagg cttcacttcg tcgtgttggc 960 cgtcttcatc tgaatctgca gcctctccaa tgtagttgcc aaacctgtgt ggtcttgatt 1020 agaatttgca caattagagc gttagattgg atgcaactta ctcatcgtag agatcatcca 1080 tcgtgcggtg ggaaaggtgg ttcaagctgc caaaaacttg tcaccagtga gctgtcgcat 1140 ctctttttgt cccaagctgc tgaagtccgt ctaatggact caccgagtgg tccgtgcgtc 1200 gaccctacca ttttggtagc cccgccgcgc ggatgccacg tccaggaggg gtccttaaat 1260 gactttgatt tctcaccacc tcttctcgtg tcagagatac ctcactgcta tcgtcggctg 1320 aataactcaa ctctttcttg tttcccttct tccccccttt aatcccacca atcttcttta 1380 ttaccggcaa gttctgtcta cacgggtgcc ctttaccaca caccgaaccc ctttaaacgc 1440 ttcacgctac agaagctttt ggtttacgac tcttctaatc agctatcgta ggccgccgat 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggtc tatgtagaat agatagtagt aggggtgtca tcctccatta 3300 tctgacaagc agataccagc atgtggccac gtgtagtgat gttgtgcaag tcattaagct 3360 gcagaggtca tgacttcaca gtgactagag ttgagtaact tgagatatgg aaagtacacg 3420 gtgtactcaa ggtacagtac aattggaggt tgacgacgtc ctttgtccac gtatcctaaa 3480 ggagcatatg gccggggggc tgaaaaatag ggcaacttca agaaagatta cctaacaggg 3540 gcttctagcc aatgaaacac atttctagta tgttcggtgg accgcttgca gaagatccat 3600 gaacactttg gggcaatttc tacggcagaa tacagggcgg tggccaaggc ggatgggtca 3660 ttctggtccc tgaaaggcga tgcaccaatc acaacgcgag gagtaacctg cagagcgatg 3720 gccgtaccag ccatgctgtc cttttctctg gcctcaagca ttttaaaaaa gctgatctct 3780 tcctctcacg tccttcgttc agcaacttct ctcttttgcc tcaactttcc cttccttccc 3840 ccttccattc cgtcgcctgt ggttgggttc tctttttctt ccttttgctt ctctttcttg 3900 aatacaggaa actgtattga agcacaaggg atttactacc aacacgtctc ccgataatca 3960 cacgcgtgcc ccttggcgaa gaccaccact cgatatacac ataggcaca atg tct tct 4018 Met Ser Ser 1 ctt ccg ccg gtc tat att gtt tcc tct gcc cgc acc cca gtc ggc tct 4066

Leu Pro Pro Val Tyr Ile Val Ser Ser Ala Arg Thr Pro Val Gly Ser

5 10 15 ttc ttg gg gtgagattgg ccttgtgcct tgtctatacg tacaatattg 4114

Phe Leu Gly 20 acttacactg actatccgct tccacag g tcg ctc tca agt ctc act gcc ccg 4166

Ser Leu Ser Ser Leu Thr Ala Pro

25 30 cag tta ggc tct cat gct att aaa g gtatgacagc tctcgagtgg 4211

Gin Leu Gly Ser His Ala Ile Lys

35 eggtaegtgt tgatatacta acaaactcga aattcag ct gcg ctc agc aaa gcg 4265 Ala Ala Leu Ser Lys Ala 40 gat gga atc aag ccg tct gat atc cag gag gtc ttc ttt ggc aat gtc 4313

Asp Gly Ile Lys Pro Ser Asp Ile Gin Glu Val Phe Phe Gly Asn Val

45 50 55 60 atc tcc gca aa gtgagtcaac gtccaccctt ataccttata gccaacatcg 4364 Ile Ser Ala Asn aaggcacact tacattctcg ttctatggat cag c gtt gga caa aat cct gct 4416 Val Gly Gin Asn Pro Ala 65 70 aga cag tgt gct ctc ggc gct ggt ctc aat gaa tca act gtc tgt act 4464

Arg Gin Cys Ala Leu Gly Ala Gly Leu Asn Glu Ser Thr Val Cys Thr

75 80 85 acg gtt aat aag gtg tgc gcg tct ggc ttg aaa gcg gtt att ctc ggt 4512

Thr Val Asn Lys Val Cys Ala Ser Gly Leu Lys Ala Val Ile Leu Gly

90 95 100 gca cag acc atc atg act ggc aat gcg gat att gtc gta gca ggc ggt 4560

Ala Gin Thr Ile Met Thr Gly Asn Ala Asp Ile Val Val Ala Gly Gly

105 110 115 gct gaa tcc atg tct aac gcc cct cat tac ctt cca aac ctt cgc gtc 4608

Ala Glu Ser Met Ser Asn Ala Pro His Tyr Leu Pro Asn Leu Arg Val

120 125 130 ggt gcg aaa tac ggc aac cag agt ctg gtg gac ggt att atg aag gat 4656

Gly Ala Lys Tyr Gly Asn Gin Ser Leu Val Asp Gly Ile Met Lys Asp

135 140 145 150 ggc ttg aca gac gca gga aag cag gaa ctc atg ggc ttg caa gcc gag 4704

Gly Leu Thr Asp Ala Gly Lys Gin Glu Leu Met Gly Leu Gin Ala Glu

155 160 165 gag tgt gct cag gat cat ggc ttt agc agg gaa caa cag gat gat tat 4752

Glu Cys Ala Gin Asp His Gly Phe Ser Arg Glu Gin Gin Asp Asp Tyr 170 175 180 gcc att cgc act tac gaa aaa gca cag gcg gct caa aag gct ggc ctt 4800

Ala Ile Arg Thr Tyr Glu Lys Ala Gln Ala Ala Gin Lys Ala Gly Leu

185 190 195 ttt gac gaa gaa att gcg cct att gaa ctt cct ggc ttt agg ggc aag 4848

Phe Asp Glu Glu Ile Ala Pro Ile Glu Leu Pro Gly Phe Arg Gly Lys

200 205 210 cca ggt gtg act gtg tca caa gac gaa gaa cca aag aat gtgaggaaga 4897

Pro Gly Val Thr Val Ser Gin Asp Glu Glu Pro Lys Asn

215 220 225 tattacttte tacagatttt ttaagtcgte ggccactgct aatatactca ttgcgtag 4955 ctt aac ccg gat aag ctt cga gct atc aag cct gca ttt atc ccc gga 5003

Leu Asn Pro Asp Lys Leu Arg Ala Ile Lys Pro Ala Phe Ile Pro Gly

230 235 240 tcc ggc acg gtc aca gcc ccg aat tcc tca cct ctt aac gac ggt gct 5051

Ser Gly Thr Val Thr Ala Pro Asn Ser Ser Pro Leu Asn Asp Gly Ala

245 250 255 gct gct gtt atc ctc gtc tca gaa gct aaa ctg aaa gag ctt aac cta 5099

Ala Ala Val Ile Leu Val Ser Glu Ala Lys Leu Lys Glu Leu Asn Leu

260 265 270 275 aag cct gtt gca aag att ctt ggc tgg gga gat gcc gcc cag cag cca 5147

Lys Pro Val Ala Lys Ile Leu Gly Trp Gly Asp Ala Ala Gin Gin Pro

280 285 290 agc aaa ttc aca act gcc cca gct cta gca att ccc aag gcc ctc agc 5195

Ser Lys Phe Thr Thr Ala Pro Ala Leu Ala Ile Pro Lys Ala Leu Ser

295 300 305 cat gca ggt gtg gct cag gat gct gtt gat gcg ttc gag att aac gaa 5243

His Ala Gly Val Ala Gin Asp Ala Val Asp Ala Phe Glu Ile Asn Glu

310 315 320 gcg ttc agc gta gtt gct ctg gcc aat atg aaa ctc ctg ggg ttg gct 5291

Ala Phe Ser Val Val Ala Leu Ala Asn Met Lys Leu Leu Gly Leu Ala

325 330 335 gaa gat aaa gtc aac atc cat ggt ggt gca gtg gct atc ggt cat cct 5339

Glu Asp Lys Val Asn Ile His Gly Gly Ala Val Ala Ile Gly His Pro

340 345 350 355 atc ggc gcc agc ggt gct cgt atc ttg act aca ttg ctc ggt gta ttg 5387 Ile Gly Ala Ser Gly Ala Arg Ile Leu Thr Thr Leu Leu Gly Val Leu

360 365 370 aaa gcg aga aag ggt aag att ggt tgt gcc ggg att tgt aat gga gga 5435

Lys Ala Arg Lys Gly Lys Ile Gly Cys Ala Gly Ile Cys Asn Gly Gly

375 380 385 ggt ggt gct agc gct att gtt gtc gaa tct ctc gtc tga gtcatgecga 5484

Gly Gly Ala Ser Ala Ile Val Val Glu Ser Leu Val

390 395 gtcgcccttg atcttggtat ggaataacgc gacgttgccc ttttctgggc ttagtcgcca 5544 tttttcgcct tgtccaggca agacactttc aatgacagtg ttgacttctt caatcacatg 5604 cttgagcttg aaataggcgt cgttgggtgg tatcttcagt tcgaggatca gccgatccat 5664 tttgttcaca accatggtca acggaaggcc ctctagcaca gcgtgcttga tgatttgctc 5724 cgtattcgat tgtactcctt ccacaatatc aacgactatc accacgccat caaccagtcg 5784 tgatgaggca gctacttcat caacaaaatt cacatgcccc ggggtatcaa gaatgttgaa 5844 caaatgagac ttccccttag tgccctgaag aactaggctc ataggagctg atttaataga 5904 aaggcctcgc tctctctcta gaaagtgaat atcggtataa cgaagctgta cttcctttct 5964 tttacctgct cgcttctcta gccgttcagt caaatcatgc gtttgcataa caagcatatc 6024 catgaacgcg gtcttcccat gatgtaaatg accgacaagc gcaatgttcc ttatttgttc 6084 aggaaaattc agaagatcag tcatgaattc ccttgaaaag tgaacccggg ggagctcagt 6144 ctcttcaatg gcgaacttct tttgctgtac gggggcaacg ataggctcgg agagaggctg 6204 tgcgtcttct tcttgaacta atgtctctac ctctgtcccg tagacctgtt gtgcgctagg 6264 gtagtattgt ttgtcttcgt gtagaatgac ggcatttgag ggggcttctg taatgagata 6324 tatcagcacc gactaaatcc aggggcttta agagagtgcc caccgtccac ctccatagac 6384 tgctgcccat ataattcttc atcttcttcc tccccaaacg cctcgttgaa ggcgaaagcc 6444 cgaggcttca cttcgtcgtg ttggccgtct tcatctgaat ctgcagcctc tccaatgtag 6504 ttgccaaacc tgtgtggtct tgattagaat ttgcacaatt agagcgttag attggatgca 6564 acttactcat cgtagagatc atccatcgtg cggtgggaaa ggtggttcaa gctgccaaaa 6624 acttgtcacc agtgagctgt cgcatctctt tttgtcccaa gctgctgaag tccgtctaat 6684 ggactcaccg agtggtccgt gcgtcgaccc taccattttg gtagccccgc cgcgcggatg 6744 ccacgtccag gaggggtcct taaatgactt tgatttctca ccacctcttc tcgtgtcaga 6804 gatacctcac tgctatcgtc ggctgaataa ctcaactctt tcttgtttcc cttcttcccc 6864 cctttaatcc caccaatctt ctttattacc ggcaagttct gtctacacgg gtgcccttta 6924 ccacacaccg aaccccttta aacgcttcac gctacagaag cttttggttt acgactcttc 6984 taatcagcta tcgtaggccg ccgatagctg attaaatatt ctagacccaa gccgctgctg 7044 gaattgacat tattatggcc gatagggttg ggcttattgc tatgtccctg aaaggatatc 7104 aaaagcaggc aaaaagccag gcataatccc cgcgtggacg gtaccctaag gataggccct 7164 aatcttatct acatgtgact gcatcgatgt gtttggtcaa aatgaggcat gtggctcacc 7224 ccacaggcgg agaaacgtgt ggctagtgca tgacagtccc ctccatagat tcaatttaat 7284 ttttcgcggc aattgtcgtg cagtttgtat ctacatttca ttccatatat caagagttag 7344 tagttggaca tcctgattat tttgtctaat tactgaaaac tcgaagtact aacctactaa 7404 taagccagtt tcaaccacta agtgctcatt tatacaatat ttgcagaacc ccgcgctacc 7464 cctccatcgc caacatgtct tccaagtcgc aattgaccta cagcgcacgc gctagcaagc 7524 accccaatgc gctcgtaaag aagctcttcg aggttgccga ggccaagaaa accaatgtca 7584 ccgtttccgc cgacgtgaca accaccaaag agctgctgga tttggctgac cgtatgcgca 7644 ccggggatgc cacttacatg tgatctagta atggttaatg gtggattata taacaggact 7704 cggtccgtac attgccgtga tcaaaactca catcgatatc ctctccgatt tcagcgaaga 7764 aaccatcacc ggtctgaagg cccttgcaga gaagcacaat ttcctcatct tcgaagatcg 7824 caagttcatc gatatcggaa acacagtcca aaagcagtac catggcggca ctctgcgtat 7884 ctctgagtgg gcccacatca tcaactgcag tattctgccc ggtgagggta tcgtcgaggc 7944 tctggcccag actgcttcgg ccgaggactt cccctacggc tccgagaggg gccttttgat 8004 ccttgcggag atgacctcca agggatcttt ggctaccggt caatatacta cttcttctgt 8064 tgactatgct cggaagtata agaagtttgt gatgggattc gtctcgacac gtcaccttgg 8124 cgaggttcag tctgaagtta gctcgccttc ggaggaggaa gattttgtcg tcttcacgac 8184 aggtgtcaac ctctcctcga agggtgacaa gctgggacag cagtaccaaa ctcctgagtc 8244 ggctgttgga cgcggtgccg actttattat tgctggccgt ggaatttatg ctgctcctga 8304 tcccgtggag gcggcgaagc agtaccagaa ggagggatgg gatgcatacc tgaagcgtgt 8364 tggtgcgcaa taagtagtgg tggatacgta ctccttttat ggcagtatgt cgcaagtatg 8424 atgcgattta taaattcagc actcgaaatg actactacta tgtgtctacg acagataccc 8484 tctccgtacg aataagacac ctgcctcgat atatggacaa attcaaaatc agggtcaagg 8544 gtcatgtttc aaagtcacaa caatctccaa catagacgag aatttgtacc ggagtgtctg 8604 aaggtgcagc tggagattgg tctattttct tagagtgggg tatcactaat gtacagtcgg 8664 tcactatcgt acaaacaatc acaattatat acaagatttc ccaccacccc ctactctaac 8724 acggcacaat tatccatcga gtcagagcct agccaccatt tggtctatgt agaatagata 8784 gtagtagggg tgtcatcctc cattatctga caagcagata ccagcatgtg gccacgtgta 8844 gtgatgttgt gcaagtcatt aagctgcaga ggtcatgact tcacagtgac tagagttgag 8904 taacttgaga tatggaaagt acacggtgta ctcaaggtac agtacaattg gaggttgacg 8964 acgtcctttg tccacgtatc ctaaaggagc atatggccgg ggggctgaaa aatagggcaa 9024 cttcaagaaa gattacctaa caggggcttc tagccaatga aacacatttc tagtatgttc 9084 ggtggaccgc ttgcagaaga tccatgaaca ctttggggca atttctacgg cagaatacag 9144 ggcggtggcc aaggcggatg ggtcattctg gtccctgaaa ggcgatgcac caatcacaac 9204 gcgaggagta acctgcagag cgatggccgt accagccatg ctgtcctttt ctctggcctc 9264 aagcatttta aaaaagctga tctcttcctc tcacgtcctt cgttcagcaa cttctctctt 9324 ttgcctcaac tttcccttcc ttcccccttc cattccgtcg cctgtggttg ggttctcttt 9384 ttcttccttt tgcttctctt tcttgaatac aggaaactgt attgaagcac aagggattta 9444 ctaccaacac gtctcccgat aatcacacgc gtgccccttg gcgaagacca ccactcgata 9504 tacacatagg cacaatgtct tctcttccgc cggtctatat tgtttcctct gcccgcaccc 9564 cagtcggctc tttcttgggg tgagattggc cttgtgcctt gtctatacgt acaatattga 9624 cttacactga ctatccgctt ccacaggtcg ctctcaagtc tcactgcccc gcagttaggc 9684 tctcatgcta ttaaaggtat gacagctctc gagtggcggt acgtgttgat atactaacaa 9744 actcgaaatt cagctgcgct cagcaaagcg gatggaatca agccgtctga tatccaggag 9804 gtcttctttg gcaatgtcat ctccgcaaag tgagtcaacg tccaccctta taccttatag 9864 ccaacatcga aggcacactt acattctcgt tctatggatc agcgttggac aaaatcctgc 9924 tagacagtgt gctctcggcg ctggtctcaa tgaatcaact gtctgtacta cggttaataa 9984 ggtgtgcgcg tctggcttga aagcggttat tctcggtgca cagaccatca tgactggcaa 10044 tgcggatatt gtcgtagcag gcggtgctga atccatgtct aacgcccctc attaccttcc 10104 aaaccttcgc gtcggtgcga aatacggcaa ccagagtctg gtggacggta ttatgaagga 10164 tggcttgaca gacgcaggaa agcaggaact catgggcttg caagccgagg agtgtgctca 10224 ggatcatggc tttagcaggg aacaacagga tgattatgcc attcgcactt acgaaaaagc 10284 acaggcggct caaaaggctg gcctttttga cgaagaaatt gcgcctattg aacttcctgg 10344 ctttaggggc aagccaggtg tgactgtgtc acaagacgaa gaaccaaaga atgtgaggaa 10404 gatattactt tctacagatt ttttaagtcg tcggccactg ctaatatact cattgcgtag 10464 cttaacccgg ataagcttcg agctatcaag cctgcattta tccccggatc cggcacggtc 10524 acagccccga attcctcacc tcttaacgac ggtgctgctg ctgttatcct cgtctcagaa 10584 gctaaactga aagagcttaa cctaaagcct gttgcaaaga ttcttggctg gggagatgcc 10644 gcccagcagc caagcaaatt cacaactgcc ccagctctag caattcccaa ggccctcagc 10704 catgcaggtg tggctcagga tgctgttgat gcgttcgaga ttaacgaagc gttcagcgta 10764 gttgctctgg ccaatatgaa actcctgggg ttggctgaag ataaagtcaa catccatggt 10824 ggtgcagtgg ctatcggtca tcctatcggc gccagcggtg ctcgtatctt gactacattg 10884 ctcggtgtat tgaaagcgag aaagggtaag attggttgtg ccgggatttg taatggagga 10944 ggtggtgcta gcgctattgt tgtcgaatct ctcgtctgag tcatgccgag tcgcccttga 11004 tcttggtatg gaat 11018

<210> 250

<211> 5509

<212> DNA

<213> synthetic <220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> bipA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5509)

<223> PFS2 essential gene

<220>

<221> exon

<222> (4010) . . (4014 )

<220>

<221> exon

<222> (4072 ) . . (4142 )

<220>

<221> exon

<222> (4229) . . (4460)

<220>

<221> exon

<222> (4534 ) . . (5509)

<400> 250 ataaaaattt cgccaccgaa gaagtatcaa ggaaactgtc tagcgagagg gactggactg 60 gaacacttga gtattcggta tccagttaag gtgatttaag ccaccccttc tcccttcaat 120 ccctaccccc cttcaaaaag gggaagaaca gtagctaaag gtagttgcca tcaagaactc 180 atacttaaaa acacgaggta agaggcccgt aatagtaatt ggttgcaaga gaaaaggggg 240 gttatccttc tgagcatcac agttttgatt acgggaagaa ccagaatatg aacgtggcaa 300 cagcggctgt cggtaccaga ccaggccttt attggatacc ggaggagtcc ctctttcgtg 360 tgtttcctac cccttttaca gtacaattac aacctacata atgtcactga tagatattta 420 tacgtgtgcc agaagatact tatgggttag tcaatgtaaa tagcttctaa ttttgattca 480 tgattgaggg gtctaaaatt ggactgaacc taatcctact caagtaggag ttagatgacg 540 atggtgagtg tgaaatgcca ggattaaggg tagcatatca gccgcacagg aactaccgag 600 ggggtggagg ctaacagccc tctttttttc tttccctttt tcctcatttt cctctctctt 660 ctcccgatga tgacgatttg tagattcata agttatagaa aaatatttgt tctatgcaca 720 cgctaatagg acatagtact gcttattgta tgtatatgcc tggtgtttaa agaagtagac 780 ttcttgagtt ccgtacggag cactatttta caatacgaat aaaaaaaatc catggagcag 840 ggaagggaag gatatagcta gctaggtccc taatagtcga tatgttattt ttgatctgtt 900 agtggagcca ctgacctgtt gaatttttgg taggtaaact tagcaacatg tccatgccat 960 aaaaggtctc caaccgagag tcacgagtga aacataccag taggtccaac aaactgtgta 1020 aggtgcagaa aaggcacatt attggccagc tccatgttga atgaatggct gcgccctgga 1080 gccggtccat agtagcttcc ctggtctctg tgtgggccga tctcacaaca taaaacatag 1140 ctcttccccc tccttccaac agattgtcga tctactcaat ttcccctttg ctatcaactt 1200 ctccccctcc tcgcttcctt ctctcactcg ccacaccgcc ctttaagaat atcacaagta 1260 atattctagc tggattatcc aaagtgtgtt ttatgttgtc cttccatggt atcttccaat 1320 tcgtttgttg acagtatatt cccttccaga gtgcactttg aagaaacccc actaccgttt 1380 acgtgacttg ctttcaatat ccggaggcaa catttgttat cgaagcatta caacgcctag 1440 tcatctcatt ctacaccttt ccacattttg actcgctctc tctcttgaca taccttaacc 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtctte acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcgge gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggtc tatgtagaat agatagtagt aggggtgtca tcctccatta 3300 tctgacaagc agataccagc atgtggccac gtgtagtgat gttgtgcaag tcattaagct 3360 gcagaggtca tgacttcaca gtgactagag ttgagtaact tgagatatgg aaagtacacg 3420 gtgtactcaa ggtacagtac aattggaggt tgacgaegte ctttgtccac gtatcctaaa 3480 ggagcatatg gccggggggc tgaaaaatag ggcaacttca agaaagatta cctaacaggg 3540 gcttctagcc aatgaaacac atttctagta tgttcggtgg accgcttgca gaagatccat 3600 gaacactttg gggcaatttc tacggcagaa tacagggcgg tggccaaggc ggatgggtca 3660 ttctggtccc tgaaaggcga tgcaccaatc acaacgcgag gagtaacctg cagagcgatg 3720 gccgtaccag ccatgctgtc cttttctctg gcctcaagca ttttaaaaaa gctgatctct 3780 tcctctcacg tccttcgttc agcaacttct ctcttttgcc tcaactttcc cttccttccc 3840 ccttccattc cgtcgcctgt ggttgggttc tctttttctt ccttttgctt ctctttcttg 3900 aatacaggaa actgtattga agcacaaggg atttactacc aacacgtctc ccgataatca 3960 cacgcgtgcc ccttggcgaa gaccaccact cgatatacac ataggcaca atg tg 4014 Met Cys 1 gtacgttccc gaagacccag gcttactgaa tgtttgatgt aactaattga cccctag c 4072 ggc atc ttc ggc tac att aac tac ctc gtc gag agg gac cgc aag ttt 4120 Gly Ile Phe Gly Tyr Ile Asn Tyr Leu Val Glu Arg Asp Arg Lys Phe 5 10 15 att ctt gac act ctt ctc aat g gttggcattc ctgttgtttt caccctcact 4172 Ile Leu Asp Thr Leu Leu Asn

20 25 ggaccttgtt tactctggtt ttgggagtgt ttgtgtattc ctctgacata ttgaag ga 4230 Gly ctc tcc cgt ctt gaa tac cga ggc tac gac tct gcg ggt ctt gcc ata 4278 Leu Ser Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala Gly Leu Ala Ile 30 35 40 gat ggc gat aag aag aat gaa gtt tgt gct ttc aag gaa gtg ggc aag 4326

Asp Gly Asp Lys Lys Asn Glu Val Cys Ala Phe Lys Glu Val Gly Lys

45 50 55 gtt gct aag ctt cga gag ctt att gat gag tgc aag cct gac ttg acc 4374 Val Ala Lys Leu Arg Glu Leu Ile Asp Glu Cys Lys Pro Asp Leu Thr

60 65 70 aaa tec ttt gaa tct cat gct ggt att tct cac act cgt tgg gcc act 4422

Lys Ser Phe Glu Ser His Ala Gly Ile Ser His Thr Arg Trp Ala Thr

75 80 85 90 cat ggg acg cct tcc cgc ttg aac tgc cac ccc cac ag gtgagtecga 4470

His Gly Thr Pro Ser Arg Leu Asn Cys His Pro His Arg

95 100 tgttccacgg tttaatcagc tcgactagga tcaggcttgt ttgctaattt tcctggcacg 4530 cag g tcc gat ctg aac tgg gaa ttt tca gtt gtc cac aat ggt atc att 4579

Ser Asp Leu Asn Trp Glu Phe Ser Val Val His Asn Gly Ile Ile

105 110 115 acc aac tac aaa gag ctc aag gct ttg ttg gag agc aag gga ttc cgc 4627

Thr Asn Tyr Lys Glu Leu Lys Ala Leu Leu Glu Ser Lys Gly Phe Arg

120 125 130 ttt gag act gac aca gac acc gaa tgt att gca aag ctt acc aaa tat 4675

Phe Glu Thr Asp Thr Asp Thr Glu Cys Ile Ala Lys Leu Thr Lys Tyr

135 140 145 150 ctc tat gac caa cag ccc gat atc gaa ttc aca gtt ttg gct aag gct 4723

Leu Tyr Asp Gin Gin Pro Asp Ile Glu Phe Thr Val Leu Ala Lys Ala

155 160 165 gtt gtg aag gag ctt gag ggc gcg ttt ggg ctt ctc atc aaa tcc gta 4771 Val Val Lys Glu Leu Glu Gly Ala Phe Gly Leu Leu Ile Lys Ser Val

170 175 180 cat tat ccc cat gag gtc att gcg gct cgc aag ggg tcg ccg ctt gtt 4819

His Tyr Pro His Glu Val Ile Ala Ala Arg Lys Gly Ser Pro Leu Val

185 190 195 att ggc gtg aga acg tcg aag aag atg aag gtg gat ttc gtg gac gtc 4867 Ile Gly Val Arg Thr Ser Lys Lys Met Lys Val Asp Phe Val Asp Val

200 205 210 gaa tac tca gaa gat gga gcc ctt cct gca gag cag gcc tcc cag aac 4915

Glu Tyr Ser Glu Asp Gly Ala Leu Pro Ala Glu Gin Ala Ser Gin Asn

215 220 225 230 gtg gcc atc aag aag tct gcg acc ggc ctc ctt gcc cca cct gac aaa 4963 Val Ala Ile Lys Lys Ser Ala Thr Gly Leu Leu Ala Pro Pro Asp Lys

235 240 245 tcc ctt ttg cat agg tca cag tct cgc gct ttc ctt tcc gat gat ggc 5011

Ser Leu Leu His Arg Ser Gin Ser Arg Ala Phe Leu Ser Asp Asp Gly

250 255 260 atc ccc cag cca gct gag ttt ttc ttg tct tct gac cca tct gca att 5059 Ile Pro Gin Pro Ala Glu Phe Phe Leu Ser Ser Asp Pro Ser Ala Ile 265 270 275 gtc gag cat acg aag aag gtc ctc tat ctc gaa gat gat gat atc gct 5107 Val Glu His Thr Lys Lys Val Leu Tyr Leu Glu Asp Asp Asp Ile Ala

280 285 290 cat gtg cat gag gga cag tta aac att cat cgc ctc aca aaa gat gat 5155

His Val His Glu Gly Gin Leu Asn Ile His Arg Leu Thr Lys Asp Asp

295 300 305 310 ggt act tct aat gtt cgc gct att cag aca att gaa ctc gaa ttg cag 5203

Gly Thr Ser Asn Val Arg Ala Ile Gin Thr Ile Glu Leu Glu Leu Gin

315 320 325 gag att atg aag ggc aag ttt gac cac ttc atg caa aag gaa att ttc 5251

Glu Ile Met Lys Gly Lys Phe Asp His Phe Met Gin Lys Glu Ile Phe

330 335 340 gag caa ccc gag tct gtg atc aat acc atg aga gga cga ctg gat gtt 5299

Glu Gin Pro Glu Ser Val Ile Asn Thr Met Arg Gly Arg Leu Asp Val

345 350 355 gca aac aag caa gtc aca ctc ggt ggc ctg egg cag tac att tct act 5347

Ala Asn Lys Gin Val Thr Leu Gly Gly Leu Arg Gin Tyr Ile Ser Thr

360 365 370 atc cgc cgc tgc aga aga atc ata ttt gtt gcc tgt gga act agt tac 5395 Ile Arg Arg Cys Arg Arg Ile Ile Phe Val Ala Cys Gly Thr Ser Tyr

375 380 385 390 cat tca tgc atg gct gtg cgt gga gtc ttt gaa gag ctt act gag att 5443

His Ser Cys Met Ala Val Arg Gly Val Phe Glu Glu Leu Thr Glu Ile

395 400 405 cct att tct gtt gaa ctt gct tec gat ttc ctg gat aga cag gct cca 5491

Pro Ile Ser Val Glu Leu Ala Ser Asp Phe Leu Asp Arg Gin Ala Pro

410 415 420 gta ttc cgt gat gac acg 5509 Val Phe Arg Asp Asp Thr

425

<210> 251

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> bipA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS <220>

<221> gene

<222> (4010) . . (5509)

<223> TUB1 essential gene

<220>

<221> exon

<222> (4010) . . (4034 )

<220>

<221> exon

<222> (4124 ) . . (4164 )

<220>

<221> exon

<222> (4239) . . (4265)

<220>

<221> exon

<222> (4337 ) . . (4412 )

<220>

<221> exon

<222> (4473) . . (5427 )

<220>

<221> exon

<222> (5481) . . (5509)

<400> 251 cgggaagatg ttccatagaa agggatcaag gatgatatcc gctgcaaccg ggggaacatt 60 ctccattagt gctttgttaa gagaaaagta gtccgccgcc gcagtaacac gaaggaaaag 120 caccgaatca gacgtaggtg cttgatcatg aaaagaggaa gatttgcgag gaacagcgtt 180 agactgaggg gaggcgagat gggcttggga atagaggcta atcgaggaga agagtgcggg 240 atcatcaatg gcttgtggaa gagtatgcgt tgtcagggta aaggcggtcg gttgctttga 300 ctacttgtca gctaattttc tttggtcgcc ttgctgggca ctggtagagg gtatatgagg 360 acgtacagtt gccaaccaac acacccgcac ctcataccgc tggccggggg tcaagttctc 420 cagaaaatac catgagtctg taccagggaa ctcgttggtc ggaaatgtag cgttaattct 480 ggttcgaaga acgggactag aggaggagag gcgatctaga cctaggtcat cgagggtagg 540 atcgaccgtg gggatgatga ggggttgcgg agcgatgaag atcgttttct cgacattggc 600 gatagcgggg aatgcgaggg tgcagagtaa gagtagatat tggagtagca tcatgacaga 660 gagttcgcta tgttcaattg acgctgctgc tagacggtca tggttctagt ccacacgcag 720 ttgagtatgg taggctatat gtatgtatgt agtaaatgag gtagatctga tactaactgt 780 tagattccta agtgaagtcc ttacatgaaa ttgattcaat gtcaatacga tcttaccata 840 aattaggcca ttgtgcgcag caatgttggt gactagaaat agagcactgt gtgtccgaga 900 cttgttctat gtgagtattt tcaatagtac ttactatcgc aagctgtacg gatagtcaaa 960 tacaatgcag ctgcacgaag agatgccgtc attgacacgt gatcgaccct tgccgtgaac 1020 taagcccaaa taagtggctg agtcagcggt gggctggaag ggcggtcacg gcatcggaca 1080 agccaatcag aaatgggatt gtgggcgacg cgacccaacg gatggatcaa caatgaaaag 1140 gtggaagtcc tgcttttggg ttgacagcgc atcgcaagac cacaacaaat tgaccgatca 1200 ttcgggtctt gggcaattgt ttaattgatc cccttagtct atttttctcc tcgtcaatca 1260 ccccaagggt ttgttctcct cccccatttc ctcccaccgg ttcccctttc cctttctttt 1320 ggttcttttc ccaggtggtg tttttttcgt ttacttaccc ctgaaacttc cttacagatc 1380 agggcatttt tccccctcta aagtccttcc acattccacc cactccgttt cacacacccc 1440 cctccttcca atcttaattc ctttgtcact ccgacattcg tcccttttag actcgacaag 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggtc tatgtagaat agatagtagt aggggtgtca tcctccatta 3300 tctgacaagc agataccagc atgtggccac gtgtagtgat gttgtgcaag tcattaagct 3360 gcagaggtca tgacttcaca gtgactagag ttgagtaact tgagatatgg aaagtacacg 3420 gtgtactcaa ggtacagtac aattggaggt tgacgaegte ctttgtccac gtatcctaaa 3480 ggagcatatg gccggggggc tgaaaaatag ggcaacttca agaaagatta cctaacaggg 3540 gcttctagcc aatgaaacac atttctagta tgttcggtgg accgcttgca gaagatccat 3600 gaacactttg gggcaatttc tacggcagaa tacagggcgg tggccaaggc ggatgggtca 3660 ttctggtccc tgaaaggcga tgcaccaatc acaacgcgag gagtaacctg cagagcgatg 3720 gccgtaccag ccatgctgtc cttttctctg gcctcaagca ttttaaaaaa gctgatctct 3780 tcctctcacg tccttcgttc agcaacttct ctcttttgcc tcaactttcc cttccttccc 3840 ccttccattc cgtcgcctgt ggttgggttc tctttttctt ccttttgctt ctctttcttg 3900 aatacaggaa actgtattga agcacaaggg atttactacc aacacgtctc ccgataatca 3960 cacgcgtgcc ccttggcgaa gaccaccact cgatatacac ataggcaca atg aga gaa 4018 Met Arg Glu 1 gtt att agt ttg aac g gtatgtgttt tctcaatctt ttcctcacac cgcctccttc 4074 Val Ile Ser Leu Asn

5 accggagtcg gtctgttgac cgttactaat tgttttttta cgtgtgcag tt ggt cag 4131 Val Gly Gin 10 gct ggt tgc cag atc gcc aat tct tgc tgg gag gtaeggagat ggaactatac 4184 Ala Gly Cys Gin Ile Ala Asn Ser Cys Trp Glu

15 20 tagaagatag aggtgtttgg aatgtgacta ttgaaactga cattatggtt ctag ctc 4241

Leu tac tgt ctt gag cac ggc atc cag gtacgtggcc acgatgacaa tccatccatc 4295 Tyr Cys Leu Glu His Gly Ile Gin

25 30 ttcatctcca tcgggcacac agactaaccg gtatacgata g ccc gat ggt tac ttg 4351 Pro Asp Gly Tyr Leu 35 acc gag gaa cgc aag aag gaa gac cct gac cat ggt ttc agc acc ttc 4399

Thr Glu Glu Arg Lys Lys Glu Asp Pro Asp His Gly Phe Ser Thr Phe

40 45 50 ttc tcc gaa act g gtaegtcgta ttgacttgtt atcttgccgc cgagccctct 4452

Phe Ser Glu Thr 55 aacaggctat ggtgtcttag gc cag ggc aag tat gtt cct cgt acc atc tac 4504

Gly Gin Gly Lys Tyr Val Pro Arg Thr Ile Tyr

60 65 gcc gat ctg gag ccc aat gtt gtc gat gag gtc cgc act ggc acc tac 4552

Ala Asp Leu Glu Pro Asn Val Val Asp Glu Val Arg Thr Gly Thr Tyr

70 75 80 cgt acc ctt ttc cac ccc gag aac atg atc acc ggc aag gag gat gcc 4600

Arg Thr Leu Phe His Pro Glu Asn Met Ile Thr Gly Lys Glu Asp Ala

85 90 95 tcg aac aac tat gcc cgt ggt cac tac acc gtt ggc aag gag atg atc 4648

Ser Asn Asn Tyr Ala Arg Gly His Tyr Thr Val Gly Lys Glu Met Ile

100 105 110 115 gac cag gtc ctc gac aag gtt cgc cgt gtg gcc gac aac tgc gct ggt 4696

Asp Gin Val Leu Asp Lys Val Arg Arg Val Ala Asp Asn Cys Ala Gly

120 125 130 ctc cag ggc ttc ctc gtc ttc cac tct ttc ggt ggt ggt act ggt tec 4744

Leu Gin Gly Phe Leu Val Phe His Ser Phe Gly Gly Gly Thr Gly Ser

135 140 145 ggt ttc ggt gct ctc ctg atg gag cgt ctg tct gtg gac tac ggc aag 4792

Gly Phe Gly Ala Leu Leu Met Glu Arg Leu Ser Val Asp Tyr Gly Lys

150 155 160 aag tec aag ctg gag ttc tgc gtc tac cct gcc ccc cag aat gcc acc 4840

Lys Ser Lys Leu Glu Phe Cys Val Tyr Pro Ala Pro Gin Asn Ala Thr

165 170 175 tec gtc gtt gag ccc tac aac tec atc ctg act acc cac acc acc ctt 4888

Ser Val Val Glu Pro Tyr Asn Ser Ile Leu Thr Thr His Thr Thr Leu

180 185 190 195 gag cac tec gac tgc agt ttc atg gtt gac aac gaa gcc atc tac gac 4936

Glu His Ser Asp Cys Ser Phe Met Val Asp Asn Glu Ala Ile Tyr Asp

200 205 210 atc tgc cgc cgc aac ctt ggc atc gag cgt ccc agc tat gag aac ctg 4984 Ile Cys Arg Arg Asn Leu Gly Ile Glu Arg Pro Ser Tyr Glu Asn Leu

215 220 225 aac cgc ctg att gct cag gtt gtc tec tec atc acc gcc tec ctg cgt 5032

Asn Arg Leu Ile Ala Gin Val Val Ser Ser Ile Thr Ala Ser Leu Arg

230 235 240 ttc gat ggt tec ctg aac gtg gat ctc aac gag ttc cag acc aac ctg 5080

Phe Asp Gly Ser Leu Asn Val Asp Leu Asn Glu Phe Gin Thr Asn Leu

245 250 255 gtt ccc tac ccc cgt att cac ttc cct ctc gtt gcc tat gct ccc gtc 5128 Val Pro Tyr Pro Arg Ile His Phe Pro Leu Val Ala Tyr Ala Pro Val

260 265 270 275 atc tcc gcg gcc aag gcc tcc cac gag gcc aac tcc gtc aac gag atc 5176 Ile Ser Ala Ala Lys Ala Ser His Glu Ala Asn Ser Val Asn Glu Ile

280 285 290 acc atg tct tgc ttc gag ccc aac aac cag atg gtc aag tgt gac ccc 5224

Thr Met Ser Cys Phe Glu Pro Asn Asn Gin Met Val Lys Cys Asp Pro

295 300 305 cgc aat ggc aag tac atg gct act tgc ttg ctg tac cgt ggt gat gtt 5272

Arg Asn Gly Lys Tyr Met Ala Thr Cys Leu Leu Tyr Arg Gly Asp Val

310 315 320 gtg ccc aag gag acc cac gcc gcc gtt gct acc ctc aag acc aag cgt 5320 Val Pro Lys Glu Thr His Ala Ala Val Ala Thr Leu Lys Thr Lys Arg

325 330 335 acc atc cag ttc gtc gac tgg tgc cct act ggt ttc aag atc ggt atc 5368

Thr Ile Gin Phe Val Asp Trp Cys Pro Thr Gly Phe Lys Ile Gly Ile

340 345 350 355 tgc tac cag ccc cct aag cag gtt ccc aac ggt gac ctt gcc aac ctc 5416

Cys Tyr Gin Pro Pro Lys Gin Val Pro Asn Gly Asp Leu Ala Asn Leu

360 365 370 agc cgt gct gt gtaagttccc tatctaccac tcgtgeaaga gataaaaagc 5467

Ser Arg Ala Val 375 tgaccagtta tag c tgc atg ctg tct aac acc acc gcc atc t 5509

Cys Met Leu Ser Asn Thr Thr Ala Ile 380

<210> 252

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> clxA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5474 )

<223> ERG10 essential gene

<220> <221> exon

<222> (4010) . . (4074 )

<220>

<221> exon

<222> (4142 ) . . (4191)

<220>

<221> exon

<222> (4249) . . (4324 )

<220>

<221> exon

<222> (4398) . . (4887 )

<220>

<221> exon

<222> (4956) . . (5474 )

<400> 252 aacgcgacgt tgcccttttc tgggcttagt cgccattttt cgccttgtcc aggcaagaca 60 ctttcaatga cagtgttgac ttcttcaatc acatgcttga gcttgaaata ggcgtcgttg 120 ggtggtatct tcagttcgag gatcagccga tccattttgt tcacaaccat ggtcaacgga 180 aggccctcta gcacagcgtg cttgatgatt tgctccgtat tcgattgtac tccttccaca 240 atatcaacga ctatcaccac gccatcaacc agtcgtgatg aggcagctac ttcatcaaca 300 aaattcacat gccccggggt atcaagaatg ttgaacaaat gagacttccc cttagtgccc 360 tgaagaacta ggctcatagg agctgattta atagaaaggc ctcgctctct ctctagaaag 420 tgaatatcgg tataacgaag ctgtacttcc tttcttttac ctgctcgctt ctctagccgt 480 tcagtcaaat catgcgtttg cataacaagc atatccatga acgcggtctt cccatgatgt 540 aaatgaccga caagcgcaat gttccttatt tgttcaggaa aattcagaag atcagtcatg 600 aattcccttg aaaagtgaac ccgggggagc tcagtctctt caatggcgaa cttcttttgc 660 tgtacggggg caacgatagg ctcggagaga ggctgtgcgt cttcttcttg aactaatgtc 720 tctacctctg tcccgtagac ctgttgtgcg ctagggtagt attgtttgtc ttcgtgtaga 780 atgacggcat ttgagggggc ttctgtaatg agatatatca gcaccgacta aatccagggg 840 ctttaagaga gtgcccaccg tccacctcca tagactgctg cccatataat tcttcatctt 900 cttcctcccc aaacgcctcg ttgaaggcga aagcccgagg cttcacttcg tcgtgttggc 960 cgtcttcatc tgaatctgca gcctctccaa tgtagttgcc aaacctgtgt ggtcttgatt 1020 agaatttgca caattagagc gttagattgg atgcaactta ctcatcgtag agatcatcca 1080 tcgtgcggtg ggaaaggtgg ttcaagctgc caaaaacttg tcaccagtga gctgtcgcat 1140 ctctttttgt cccaagctgc tgaagtccgt ctaatggact caccgagtgg tccgtgcgtc 1200 gaccctacca ttttggtagc cccgccgcgc ggatgccacg tccaggaggg gtccttaaat 1260 gactttgatt tctcaccacc tcttctcgtg tcagagatac ctcactgcta tcgtcggctg 1320 aataactcaa ctctttcttg tttcccttct tccccccttt aatcccacca atcttcttta 1380 ttaccggcaa gttctgtcta cacgggtgcc ctttaccaca caccgaaccc ctttaaacgc 1440 ttcacgctac agaagctttt ggtttacgac tcttctaatc agctatcgta ggccgccgat 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggta gtgtggtctg aagttttgca aagtgagtga ggaggggaaa 3300 agaaegtcgg cggggagagg ggcaacgtga agaactccat tttttttttt tctggatatg 3360 gataattaac tagtagcggg aagtagatcg cgcaatcggt atttgeaagg atagtatgea 3420 attacttcga agattaagaa aggaacaaga ggaaaaatag aaaagaattt atttatattc 3480 atttgaaatt tacattgact ggattgaatt gtcttatttg aattgggtga ctagcatgtt 3540 tgggagaagt gtactaatat gaatgaatgg tatggagtac tacaaataac atacagggac 3600 gctaagaeeg tgacaaccaa agagagaaaa caaaaaaagc aataaaaegg ttctttagaa 3660 tcaatggaca ctggacagaa cattggagaa tctgccacta atcctgtttg ggtcggtgga 3720 actttacccc aaggggcaag cggcagcgga agaggccaga ttgaacttaa gctaccttcc 3780 cagcttcata taatcaacct ccccaacact cgtcttctct ttctcctctc ctctctcttc 3840 ttccttctgt cgtgcggtta gcttgtggta cgctctcacc ttgacactct tttccccctc 3900 gaacctctac ggaggaccaa ttgcttgatc gtttgcttat ttgtttcttt ctttcctaaa 3960 gctactaccg agtcgggctt cttagtttga gagcccgtta tcattcatc atg tct tct 4018 Met Ser Ser 1 ctt ccg ccg gtc tat att gtt tcc tct gcc cgc acc cca gtc ggc tct 4066 Leu Pro Pro Val Tyr Ile Val Ser Ser Ala Arg Thr Pro Val Gly Ser 5 10 15 ttc ttg gg gtgagattgg ccttgtgcct tgtctatacg tacaatattg 4114 Phe Leu Gly 20 acttacactg actatccgct tccacag g tcg ctc tca agt ctc act gcc ccg 4166 Ser Leu Ser Ser Leu Thr Ala Pro 25 30 cag tta ggc tct cat gct att aaa g gtatgacagc tctcgagtgg 4211 Gin Leu Gly Ser His Ala Ile Lys

35 eggtaegtgt tgatatacta acaaactcga aattcag ct gcg ctc agc aaa gcg 4265 Ala Ala Leu Ser Lys Ala 40 gat gga atc aag ccg tct gat atc cag gag gtc ttc ttt ggc aat gtc 4313 Asp Gly Ile Lys Pro Ser Asp Ile Gin Glu Val Phe Phe Gly Asn Val 45 50 55 60 atc tcc gca aa gtgagtcaac gtccaccctt ataccttata gccaacatcg 4364 Ile Ser Ala Asn aaggcacact tacattctcg ttctatggat cag c gtt gga caa aat cct gct 4416 Val Gly Gin Asn Pro Ala 65 70 aga cag tgt gct ctc ggc gct ggt ctc aat gaa tca act gtc tgt act 4464 Arg Gin Cys Ala Leu Gly Ala Gly Leu Asn Glu Ser Thr Val Cys Thr 75 80 85 acg gtt aat aag gtg tgc gcg tct ggc ttg aaa gcg gtt att ctc ggt 4512

Thr Val Asn Lys Val Cys Ala Ser Gly Leu Lys Ala Val Ile Leu Gly

90 95 100 gca cag acc atc atg act ggc aat gcg gat att gtc gta gca ggc ggt 4560

Ala Gin Thr Ile Met Thr Gly Asn Ala Asp Ile Val Val Ala Gly Gly

105 110 115 gct gaa tec atg tct aac gcc cct cat tac ctt cca aac ctt cgc gtc 4608

Ala Glu Ser Met Ser Asn Ala Pro His Tyr Leu Pro Asn Leu Arg Val

120 125 130 ggt gcg aaa tac ggc aac cag agt ctg gtg gac ggt att atg aag gat 4656

Gly Ala Lys Tyr Gly Asn Gin Ser Leu Val Asp Gly Ile Met Lys Asp

135 140 145 150 ggc ttg aca gac gca gga aag cag gaa ctc atg ggc ttg caa gcc gag 4704

Gly Leu Thr Asp Ala Gly Lys Gin Glu Leu Met Gly Leu Gin Ala Glu

155 160 165 gag tgt gct cag gat cat ggc ttt agc agg gaa caa cag gat gat tat 4752

Glu Cys Ala Gin Asp His Gly Phe Ser Arg Glu Gin Gin Asp Asp Tyr

170 175 180 gcc att cgc act tac gaa aaa gca cag gcg gct caa aag gct ggc ctt 4800

Ala Ile Arg Thr Tyr Glu Lys Ala Gin Ala Ala Gin Lys Ala Gly Leu

185 190 195 ttt gac gaa gaa att gcg cct att gaa ctt cct ggc ttt agg ggc aag 4848

Phe Asp Glu Glu Ile Ala Pro Ile Glu Leu Pro Gly Phe Arg Gly Lys

200 205 210 cca ggt gtg act gtg tca caa gac gaa gaa cca aag aat gtgaggaaga 4897

Pro Gly Val Thr Val Ser Gin Asp Glu Glu Pro Lys Asn

215 220 225 tattacttte tacagatttt ttaagtcgte ggccactgct aatatactca ttgcgtag 4955 ctt aac ccg gat aag ctt cga gct atc aag cct gca ttt atc ccc gga 5003

Leu Asn Pro Asp Lys Leu Arg Ala Ile Lys Pro Ala Phe Ile Pro Gly

230 235 240 tcc ggc acg gtc aca gcc ccg aat tcc tca cct ctt aac gac ggt gct 5051

Ser Gly Thr Val Thr Ala Pro Asn Ser Ser Pro Leu Asn Asp Gly Ala

245 250 255 gct gct gtt atc ctc gtc tca gaa gct aaa ctg aaa gag ctt aac cta 5099

Ala Ala Val Ile Leu Val Ser Glu Ala Lys Leu Lys Glu Leu Asn Leu

260 265 270 275 aag cct gtt gca aag att ctt ggc tgg gga gat gcc gcc cag cag cca 5147

Lys Pro Val Ala Lys Ile Leu Gly Trp Gly Asp Ala Ala Gin Gin Pro

280 285 290 agc aaa ttc aca act gcc cca gct cta gca att ccc aag gcc ctc agc 5195

Ser Lys Phe Thr Thr Ala Pro Ala Leu Ala Ile Pro Lys Ala Leu Ser

295 300 305 cat gca ggt gtg gct cag gat gct gtt gat gcg ttc gag att aac gaa 5243

His Ala Gly Val Ala Gin Asp Ala Val Asp Ala Phe Glu Ile Asn Glu 310 315 320 gcg ttc agc gta gtt gct ctg gcc aat atg aaa ctc ctg ggg ttg gct 5291

Ala Phe Ser Val Val Ala Leu Ala Asn Met Lys Leu Leu Gly Leu Ala

325 330 335 gaa gat aaa gtc aac atc cat ggt ggt gca gtg gct atc ggt cat cct 5339

Glu Asp Lys Val Asn Ile His Gly Gly Ala Val Ala Ile Gly His Pro

340 345 350 355 atc ggc gcc agc ggt gct cgt atc ttg act aca ttg ctc ggt gta ttg 5387 Ile Gly Ala Ser Gly Ala Arg Ile Leu Thr Thr Leu Leu Gly Val Leu

360 365 370 aaa gcg aga aag ggt aag att ggt tgt gcc ggg att tgt aat gga gga 5435

Lys Ala Arg Lys Gly Lys Ile Gly Cys Ala Gly Ile Cys Asn Gly Gly

375 380 385 ggt ggt gct agc gct att gtt gtc gaa tct ctc gtc tga gtcatgecga 5484

Gly Gly Ala Ser Ala Ile Val Val Glu Ser Leu Val

390 395 gtcgcccttg atcttggtat ggaat 5509

<210> 253

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> clxA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5509)

<223> FKS2 essential gene

<220>

<221> exon

<222> (4010) . . (4014 )

<220>

<221> exon

<222> (4072 ) . . (4142 )

<220>

<221> exon

<222> (4229) . . (4460) <220>

<221> exon

<222> (4534 ) . . (5509)

<400> 253 ataaaaattt cgccaccgaa gaagtatcaa ggaaactgtc tagcgagagg gactggactg 60 gaacacttga gtattcggta tccagttaag gtgatttaag ccaccccttc tcccttcaat 120 ccctaccccc cttcaaaaag gggaagaaca gtagctaaag gtagttgcca tcaagaactc 180 atacttaaaa acacgaggta agaggcccgt aatagtaatt ggttgcaaga gaaaaggggg 240 gttatccttc tgagcatcac agttttgatt acgggaagaa ccagaatatg aacgtggcaa 300 cagcggctgt cggtaccaga ccaggccttt attggatacc ggaggagtcc ctctttcgtg 360 tgtttcctac cccttttaca gtacaattac aacctacata atgtcactga tagatattta 420 tacgtgtgcc agaagatact tatgggttag tcaatgtaaa tagcttctaa ttttgattca 480 tgattgaggg gtctaaaatt ggactgaacc taatcctact caagtaggag ttagatgacg 540 atggtgagtg tgaaatgcca ggattaaggg tagcatatca gccgcacagg aactaccgag 600 ggggtggagg ctaacagccc tctttttttc tttccctttt tcctcatttt cctctctctt 660 ctcccgatga tgacgatttg tagattcata agttatagaa aaatatttgt tctatgcaca 720 cgctaatagg acatagtact gcttattgta tgtatatgcc tggtgtttaa agaagtagac 780 ttcttgagtt ccgtacggag cactatttta caatacgaat aaaaaaaatc catggagcag 840 ggaagggaag gatatagcta gctaggtccc taatagtcga tatgttattt ttgatctgtt 900 agtggagcca ctgacctgtt gaatttttgg taggtaaact tagcaacatg tccatgccat 960 aaaaggtctc caaccgagag tcacgagtga aacataccag taggtccaac aaactgtgta 1020 aggtgcagaa aaggcacatt attggccagc tccatgttga atgaatggct gcgccctgga 1080 gccggtccat agtagcttcc ctggtctctg tgtgggccga tctcacaaca taaaacatag 1140 ctcttccccc tccttccaac agattgtcga tctactcaat ttcccctttg ctatcaactt 1200 ctccccctcc tcgcttcctt ctctcactcg ccacaccgcc ctttaagaat atcacaagta 1260 atattctagc tggattatcc aaagtgtgtt ttatgttgtc cttccatggt atcttccaat 1320 tcgtttgttg acagtatatt cccttccaga gtgcactttg aagaaacccc actaccgttt 1380 acgtgacttg ctttcaatat ccggaggcaa catttgttat cgaagcatta caacgcctag 1440 tcatctcatt ctacaccttt ccacattttg actcgctctc tctcttgaca taccttaacc 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggta gtgtggtctg aagttttgca aagtgagtga ggaggggaaa 3300 agaacgtcgg cggggagagg ggcaacgtga agaactccat tttttttttt tctggatatg 3360 gataattaac tagtagcggg aagtagatcg cgcaatcggt atttgcaagg atagtatgca 3420 attacttcga agattaagaa aggaacaaga ggaaaaatag aaaagaattt atttatattc 3480 atttgaaatt tacattgact ggattgaatt gtcttatttg aattgggtga ctagcatgtt 3540 tgggagaagt gtactaatat gaatgaatgg tatggagtac tacaaataac atacagggac 3600 gctaagaccg tgacaaccaa agagagaaaa caaaaaaagc aataaaacgg ttctttagaa 3660 tcaatggaca ctggacagaa cattggagaa tctgccacta atcctgtttg ggtcggtgga 3720 actttacccc aaggggcaag cggcagcgga agaggccaga ttgaacttaa gctaccttcc 3780 cagcttcata taatcaacct ccccaacact cgtcttctct ttctcctctc ctctctcttc 3840 ttccttctgt cgtgcggtta gcttgtggta cgctctcacc ttgacactct tttccccctc 3900 gaacctctac ggaggaccaa ttgcttgatc gtttgcttat ttgtttcttt ctttcctaaa 3960 gctactaccg agtcgggctt cttagtttga gagcccgtta tcattcatc atg tg 4014 Met Cys 1 gtacgttccc gaagacccag gcttactgaa tgtttgatgt aactaattga cccctag c 4072 ggc atc ttc ggc tac att aac tac ctc gtc gag agg gac cgc aag ttt 4120 Gly Ile Phe Gly Tyr Ile Asn Tyr Leu Val Glu Arg Asp Arg Lys Phe 5 10 15 att ctt gac act ctt ctc aat g gttggcattc ctgttgtttt caccctcact 4172 Ile Leu Asp Thr Leu Leu Asn

20 25 ggaccttgtt tactctggtt ttgggagtgt ttgtgtattc ctctgacata ttgaag ga 4230 Gly ctc tcc cgt ctt gaa tac cga ggc tac gac tct gcg ggt ctt gcc ata 4278 Leu Ser Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala Gly Leu Ala Ile 30 35 40 gat ggc gat aag aag aat gaa gtt tgt gct ttc aag gaa gtg ggc aag 4326 Asp Gly Asp Lys Lys Asn Glu Val Cys Ala Phe Lys Glu Val Gly Lys 45 50 55 gtt gct aag ctt cga gag ctt att gat gag tgc aag cct gac ttg acc 4374 Val Ala Lys Leu Arg Glu Leu Ile Asp Glu Cys Lys Pro Asp Leu Thr 60 65 70 aaa tcc ttt gaa tct cat gct ggt att tct cac act cgt tgg gcc act 4422 Lys Ser Phe Glu Ser His Ala Gly Ile Ser His Thr Arg Trp Ala Thr 75 80 85 90 cat ggg acg cct tcc cgc ttg aac tgc cac ccc cac ag gtgagtecga 4470 His Gly Thr Pro Ser Arg Leu Asn Cys His Pro His Arg 95 100 tgttccacgg tttaatcagc tcgactagga tcaggcttgt ttgctaattt tcctggcacg 4530 cag g tcc gat ctg aac tgg gaa ttt tca gtt gtc cac aat ggt atc att 4579 Ser Asp Leu Asn Trp Glu Phe Ser Val Val His Asn Gly Ile Ile 105 110 115 acc aac tac aaa gag ctc aag gct ttg ttg gag agc aag gga ttc cgc 4627 Thr Asn Tyr Lys Glu Leu Lys Ala Leu Leu Glu Ser Lys Gly Phe Arg 120 125 130 ttt gag act gac aca gac acc gaa tgt att gca aag ctt acc aaa tat 4675 Phe Glu Thr Asp Thr Asp Thr Glu Cys Ile Ala Lys Leu Thr Lys Tyr 135 140 145 150 ctc tat gac caa cag ccc gat atc gaa ttc aca gtt ttg gct aag gct 4723

Leu Tyr Asp Gin Gin Pro Asp Ile Glu Phe Thr Val Leu Ala Lys Ala

155 160 165 gtt gtg aag gag ctt gag ggc gcg ttt ggg ctt ctc atc aaa tcc gta 4771 Val Val Lys Glu Leu Glu Gly Ala Phe Gly Leu Leu Ile Lys Ser Val

170 175 180 cat tat ccc cat gag gtc att gcg gct cgc aag ggg tcg ccg ctt gtt 4819

His Tyr Pro His Glu Val Ile Ala Ala Arg Lys Gly Ser Pro Leu Val

185 190 195 att ggc gtg aga acg tcg aag aag atg aag gtg gat ttc gtg gac gtc 4867 Ile Gly Val Arg Thr Ser Lys Lys Met Lys Val Asp Phe Val Asp Val

200 205 210 gaa tac tca gaa gat gga gcc ctt cct gca gag cag gcc tcc cag aac 4915

Glu Tyr Ser Glu Asp Gly Ala Leu Pro Ala Glu Gin Ala Ser Gin Asn

215 220 225 230 gtg gcc atc aag aag tct gcg acc ggc ctc ctt gcc cca cct gac aaa 4963 Val Ala Ile Lys Lys Ser Ala Thr Gly Leu Leu Ala Pro Pro Asp Lys

235 240 245 tcc ctt ttg cat agg tca cag tct cgc gct ttc ctt tcc gat gat ggc 5011

Ser Leu Leu His Arg Ser Gin Ser Arg Ala Phe Leu Ser Asp Asp Gly

250 255 260 atc ccc cag cca gct gag ttt ttc ttg tct tct gac cca tct gca att 5059 Ile Pro Gin Pro Ala Glu Phe Phe Leu Ser Ser Asp Pro Ser Ala Ile

265 270 275 gtc gag cat acg aag aag gtc ctc tat ctc gaa gat gat gat atc gct 5107 Val Glu His Thr Lys Lys Val Leu Tyr Leu Glu Asp Asp Asp Ile Ala

280 285 290 cat gtg cat gag gga cag tta aac att cat cgc ctc aca aaa gat gat 5155

His Val His Glu Gly Gin Leu Asn Ile His Arg Leu Thr Lys Asp Asp

295 300 305 310 ggt act tct aat gtt cgc gct att cag aca att gaa ctc gaa ttg cag 5203

Gly Thr Ser Asn Val Arg Ala Ile Gin Thr Ile Glu Leu Glu Leu Gin

315 320 325 gag att atg aag ggc aag ttt gac cac ttc atg caa aag gaa att ttc 5251

Glu Ile Met Lys Gly Lys Phe Asp His Phe Met Gin Lys Glu Ile Phe

330 335 340 gag caa ccc gag tct gtg atc aat acc atg aga gga cga ctg gat gtt 5299

Glu Gin Pro Glu Ser Val Ile Asn Thr Met Arg Gly Arg Leu Asp Val

345 350 355 gca aac aag caa gtc aca ctc ggt ggc ctg egg cag tac att tct act 5347

Ala Asn Lys Gin Val Thr Leu Gly Gly Leu Arg Gin Tyr Ile Ser Thr

360 365 370 atc cgc cgc tgc aga aga atc ata ttt gtt gcc tgt gga act agt tac 5395 Ile Arg Arg Cys Arg Arg Ile Ile Phe Val Ala Cys Gly Thr Ser Tyr

375 380 385 390 cat tca tgc atg gct gtg cgt gga gtc ttt gaa gag ctt act gag att 5443

His Ser Cys Met Ala Val Arg Gly Val Phe Glu Glu Leu Thr Glu Ile

395 400 405 cct att tct gtt gaa ctt gct tcc gat ttc ctg gat aga cag gct cca 5491 Pro Ile Ser Val Glu Leu Ala Ser Asp Phe Leu Asp Arg Gin Ala Pro 410 415 420 gta ttc cgt gat gac acg 5509 Val Phe Arg Asp Asp Thr 425

<210> 254

<211> 5509

<212> DNA

<213> synthetic

<220>

<221> gene

<222> (1970) . . (2868)

<223> pyrG

<220>

<221> promoter

<222> (3260) . . (4009)

<223> clxA promoter

<220>

<221> promoter

<222> (4004 ) . . (4009)

<223> TIS

<220>

<221> gene

<222> (4010) . . (5509)

<223> TUB1 essential gene

<220>

<221> exon

<222> (4010) . . (4034 )

<220>

<221> exon

<222> (4124 ) . . (4164 )

<220>

<221> exon

<222> (4239) . . (4265)

<220>

<221> exon

<222> (4337 ) . . (4412 )

<220>

<221> exon

<222> (4473) . . (5427 )

<220>

<221> exon

<222> (5481) . . (5509)

<400> 254 cgggaagatg ttccatagaa agggatcaag gatgatatcc gctgcaaccg ggggaacatt 60 ctccattagt gctttgttaa gagaaaagta gtccgccgcc gcagtaacac gaaggaaaag 120 caccgaatca gacgtaggtg cttgatcatg aaaagaggaa gatttgcgag gaacagcgtt 180 agactgaggg gaggcgagat gggcttggga atagaggcta atcgaggaga agagtgcggg 240 atcatcaatg gcttgtggaa gagtatgcgt tgtcagggta aaggcggtcg gttgctttga 300 ctacttgtca gctaattttc tttggtcgcc ttgctgggca ctggtagagg gtatatgagg 360 acgtacagtt gccaaccaac acacccgcac ctcataccgc tggccggggg tcaagttctc 420 cagaaaatac catgagtctg taccagggaa ctcgttggtc ggaaatgtag cgttaattct 480 ggttcgaaga acgggactag aggaggagag gcgatctaga cctaggtcat cgagggtagg 540 atcgaccgtg gggatgatga ggggttgcgg agcgatgaag atcgttttct cgacattggc 600 gatagcgggg aatgcgaggg tgcagagtaa gagtagatat tggagtagca tcatgacaga 660 gagttcgcta tgttcaattg acgctgctgc tagacggtca tggttctagt ccacacgcag 720 ttgagtatgg taggctatat gtatgtatgt agtaaatgag gtagatctga tactaactgt 780 tagattccta agtgaagtcc ttacatgaaa ttgattcaat gtcaatacga tcttaccata 840 aattaggcca ttgtgcgcag caatgttggt gactagaaat agagcactgt gtgtccgaga 900 cttgttctat gtgagtattt tcaatagtac ttactatcgc aagctgtacg gatagtcaaa 960 tacaatgcag ctgcacgaag agatgccgtc attgacacgt gatcgaccct tgccgtgaac 1020 taagcccaaa taagtggctg agtcagcggt gggctggaag ggcggtcacg gcatcggaca 1080 agccaatcag aaatgggatt gtgggcgacg cgacccaacg gatggatcaa caatgaaaag 1140 gtggaagtcc tgcttttggg ttgacagcgc atcgcaagac cacaacaaat tgaccgatca 1200 ttcgggtctt gggcaattgt ttaattgatc cccttagtct atttttctcc tcgtcaatca 1260 ccccaagggt ttgttctcct cccccatttc ctcccaccgg ttcccctttc cctttctttt 1320 ggttcttttc ccaggtggtg tttttttcgt ttacttaccc ctgaaacttc cttacagatc 1380 agggcatttt tccccctcta aagtccttcc acattccacc cactccgttt cacacacccc 1440 cctccttcca atcttaattc ctttgtcact ccgacattcg tcccttttag actcgacaag 1500 agctgattaa atattctaga cccaagccgc tgctggaatt gacattatta tggccgatag 1560 ggttgggctt attgctatgt ccctgaaagg atatcaaaag caggcaaaaa gccaggcata 1620 atccccgcgt ggacggtacc ctaaggatag gccctaatct tatctacatg tgactgcatc 1680 gatgtgtttg gtcaaaatga ggcatgtggc tcaccccaca ggcggagaaa cgtgtggcta 1740 gtgcatgaca gtcccctcca tagattcaat ttaatttttc gcggcaattg tcgtgcagtt 1800 tgtatctaca tttcattcca tatatcaaga gttagtagtt ggacatcctg attattttgt 1860 ctaattactg aaaactcgaa gtactaacct actaataagc cagtttcaac cactaagtgc 1920 tcatttatac aatatttgca gaaccccgcg ctacccctcc atcgccaaca tgtcttccaa 1980 gtcgcaattg acctacagcg cacgcgctag caagcacccc aatgcgctcg taaagaagct 2040 cttcgaggtt gccgaggcca agaaaaccaa tgtcaccgtt tccgccgacg tgacaaccac 2100 caaagagctg ctggatttgg ctgaccgtat gcgcaccggg gatgccactt acatgtgatc 2160 tagtaatggt taatggtgga ttatataaca ggactcggtc cgtacattgc cgtgatcaaa 2220 actcacatcg atatcctctc cgatttcagc gaagaaacca tcaccggtct gaaggccctt 2280 gcagagaagc acaatttcct catcttcgaa gatcgcaagt tcatcgatat cggaaacaca 2340 gtccaaaagc agtaccatgg cggcactctg cgtatctctg agtgggccca catcatcaac 2400 tgcagtattc tgcccggtga gggtatcgtc gaggctctgg cccagactgc ttcggccgag 2460 gacttcccct acggctccga gaggggcctt ttgatccttg cggagatgac ctccaaggga 2520 tctttggcta ccggtcaata tactacttct tctgttgact atgctcggaa gtataagaag 2580 tttgtgatgg gattcgtctc gacacgtcac cttggcgagg ttcagtctga agttagctcg 2640 ccttcggagg aggaagattt tgtcgtcttc acgacaggtg tcaacctctc ctcgaagggt 2700 gacaagctgg gacagcagta ccaaactcct gagtcggctg ttggacgcgg tgccgacttt 2760 attattgctg gccgtggaat ttatgctgct cctgatcccg tggaggcggc gaagcagtac 2820 cagaaggagg gatgggatgc atacctgaag cgtgttggtg cgcaataagt agtggtggat 2880 acgtactcct tttatggcag tatgtcgcaa gtatgatgcg atttataaat tcagcactcg 2940 aaatgactac tactatgtgt ctacgacaga taccctctcc gtacgaataa gacacctgcc 3000 tcgatatatg gacaaattca aaatcagggt caagggtcat gtttcaaagt cacaacaatc 3060 tccaacatag acgagaattt gtaccggagt gtctgaaggt gcagctggag attggtctat 3120 tttcttagag tggggtatca ctaatgtaca gtcggtcact atcgtacaaa caatcacaat 3180 tatatacaag atttcccacc accccctact ctaacacggc acaattatcc atcgagtcag 3240 agcctagcca ccatttggta gtgtggtctg aagttttgca aagtgagtga ggaggggaaa 3300 agaacgtcgg cggggagagg ggcaacgtga agaactccat tttttttttt tctggatatg 3360 gataattaac tagtagcggg aagtagatcg cgcaatcggt atttgcaagg atagtatgca 3420 attacttcga agattaagaa aggaacaaga ggaaaaatag aaaagaattt atttatattc 3480 atttgaaatt tacattgact ggattgaatt gtcttatttg aattgggtga ctagcatgtt 3540 tgggagaagt gtactaatat gaatgaatgg tatggagtac tacaaataac atacagggac 3600 gctaagaccg tgacaaccaa agagagaaaa caaaaaaagc aataaaacgg ttctttagaa 3660 tcaatggaca ctggacagaa cattggagaa tctgccacta atcctgtttg ggtcggtgga 3720 actttacccc aaggggcaag cggcagcgga agaggccaga ttgaacttaa gctaccttcc 3780 cagcttcata taatcaacct ccccaacact cgtcttctct ttctcctctc ctctctcttc 3840 ttccttctgt cgtgcggtta gcttgtggta cgctctcacc ttgacactct tttccccctc 3900 gaacctctac ggaggaccaa ttgcttgatc gtttgcttat ttgtttcttt ctttcctaaa 3960 gctactaccg agtcgggctt cttagtttga gagcccgtta tcattcatc atg aga gaa 4018 Met Arg Glu 1 gtt att agt ttg aac g gtatgtgttt tctcaatctt ttcctcacac cgcctccttc 4074 Val Ile Ser Leu Asn

5 accggagtcg gtctgttgac cgttactaat tgttttttta cgtgtgcag tt ggt cag 4131 Val Gly Gin 10 gct ggt tgc cag atc gcc aat tct tgc tgg gag gtaeggagat ggaactatac 4184 Ala Gly Cys Gin Ile Ala Asn Ser Cys Trp Glu

15 20 tagaagatag aggtgtttgg aatgtgacta ttgaaactga cattatggtt ctag ctc 4241

Leu tac tgt ctt gag cac ggc atc cag gtacgtggcc acgatgacaa tccatccatc 4295 Tyr Cys Leu Glu His Gly Ile Gin

25 30 ttcatctcca tcgggcacac agactaaccg gtatacgata g ccc gat ggt tac ttg 4351 Pro Asp Gly Tyr Leu 35 acc gag gaa cgc aag aag gaa gac cct gac cat ggt ttc agc acc ttc 4399 Thr Glu Glu Arg Lys Lys Glu Asp Pro Asp His Gly Phe Ser Thr Phe 40 45 50 ttc tcc gaa act g gtacgtcgta ttgacttgtt atcttgccgc cgagccctct 4452 Phe Ser Glu Thr

55 aacaggctat ggtgtcttag gc cag ggc aag tat gtt cct cgt acc atc tac 4504 Gly Gin Gly Lys Tyr Val Pro Arg Thr Ile Tyr 60 65 gcc gat ctg gag ccc aat gtt gtc gat gag gtc cgc act ggc acc tac 4552 Ala Asp Leu Glu Pro Asn Val Val Asp Glu Val Arg Thr Gly Thr Tyr 70 75 80 cgt acc ctt ttc cac ccc gag aac atg atc acc ggc aag gag gat gcc 4600 Arg Thr Leu Phe His Pro Glu Asn Met Ile Thr Gly Lys Glu Asp Ala 85 90 95 tcg aac aac tat gcc cgt ggt cac tac acc gtt ggc aag gag atg atc 4648 Ser Asn Asn Tyr Ala Arg Gly His Tyr Thr Val Gly Lys Glu Met Ile 100 105 110 115 gac cag gtc ctc gac aag gtt cgc cgt gtg gcc gac aac tgc gct ggt 4696 Asp Gin Val Leu Asp Lys Val Arg Arg Val Ala Asp Asn Cys Ala Gly 120 125 130 ctc cag ggc ttc ctc gtc ttc cac tct ttc ggt ggt ggt act ggt tcc 4744 Leu Gin Gly Phe Leu Val Phe His Ser Phe Gly Gly Gly Thr Gly Ser 135 140 145 ggt ttc ggt gct ctc ctg atg gag cgt ctg tct gtg gac tac ggc aag 4792

Gly Phe Gly Ala Leu Leu Met Glu Arg Leu Ser Val Asp Tyr Gly Lys

150 155 160 aag tcc aag ctg gag ttc tgc gtc tac cct gcc ccc cag aat gcc acc 4840

Lys Ser Lys Leu Glu Phe Cys Val Tyr Pro Ala Pro Gin Asn Ala Thr

165 170 175 tcc gtc gtt gag ccc tac aac tcc atc ctg act acc cac acc acc ctt 4888

Ser Val Val Glu Pro Tyr Asn Ser Ile Leu Thr Thr His Thr Thr Leu

180 185 190 195 gag cac tcc gac tgc agt ttc atg gtt gac aac gaa gcc atc tac gac 4936

Glu His Ser Asp Cys Ser Phe Met Val Asp Asn Glu Ala Ile Tyr Asp

200 205 210 atc tgc cgc cgc aac ctt ggc atc gag cgt ccc agc tat gag aac ctg 4984 Ile Cys Arg Arg Asn Leu Gly Ile Glu Arg Pro Ser Tyr Glu Asn Leu

215 220 225 aac cgc ctg att gct cag gtt gtc tcc tcc atc acc gcc tcc ctg cgt 5032

Asn Arg Leu Ile Ala Gin Val Val Ser Ser Ile Thr Ala Ser Leu Arg

230 235 240 ttc gat ggt tcc ctg aac gtg gat ctc aac gag ttc cag acc aac ctg 5080

Phe Asp Gly Ser Leu Asn Val Asp Leu Asn Glu Phe Gin Thr Asn Leu

245 250 255 gtt ccc tac ccc cgt att cac ttc cct ctc gtt gcc tat gct ccc gtc 5128 Val Pro Tyr Pro Arg Ile His Phe Pro Leu Val Ala Tyr Ala Pro Val

260 265 270 275 atc tcc gcg gcc aag gcc tcc cac gag gcc aac tcc gtc aac gag atc 5176 Ile Ser Ala Ala Lys Ala Ser His Glu Ala Asn Ser Val Asn Glu Ile

280 285 290 acc atg tct tgc ttc gag ccc aac aac cag atg gtc aag tgt gac ccc 5224

Thr Met Ser Cys Phe Glu Pro Asn Asn Gin Met Val Lys Cys Asp Pro

295 300 305 cgc aat ggc aag tac atg gct act tgc ttg ctg tac cgt ggt gat gtt 5272

Arg Asn Gly Lys Tyr Met Ala Thr Cys Leu Leu Tyr Arg Gly Asp Val

310 315 320 gtg ccc aag gag acc cac gcc gcc gtt gct acc ctc aag acc aag cgt 5320 Val Pro Lys Glu Thr His Ala Ala Val Ala Thr Leu Lys Thr Lys Arg

325 330 335 acc atc cag ttc gtc gac tgg tgc cct act ggt ttc aag atc ggt atc 5368

Thr Ile Gin Phe Val Asp Trp Cys Pro Thr Gly Phe Lys Ile Gly Ile

340 345 350 355 tgc tac cag ccc cct aag cag gtt ccc aac ggt gac ctt gcc aac ctc 5416

Cys Tyr Gin Pro Pro Lys Gin Val Pro Asn Gly Asp Leu Ala Asn Leu

360 365 370 agc cgt gct gt gtaagttccc tatctaccac tcgtgeaaga gataaaaagc 5467

Ser Arg Ala Val

375 tgaccagtta tag c tgc atg ctg tct aac acc acc gcc atc t 5509

Cys Met Leu Ser Asn Thr Thr Ala Ile 380

Claims (16)

1. A microbial production cell genetically engineered to synthesize a product, said microbial cell further comprising: a. a first essential gene operably linked to a first burden-sensing promoter, and b. a second essential gene operatively linked to a second burden-sensing promoter, wherein said first burden-sensing promoter is heterologous with respect to said first essential gene, and said second burden-sensing promoter is heterologous with respect to said second essential gene; wherein synthesis of the product confers a burden on said cell, and wherein expression of said first essential genes is up-regulated when said first burden-sensing promoter is induced by said burden relative to a basal level expression of said first essential gene when said first burden-sensing promoter is not induced, and expression of said second essential gene is up-regulated when said second burden-sensing promoter is induced by said burden relative to a basal level expression of said second essential gene when said second burden-sensing promoter is not induced.

2. The microbial production cell according to claim 1, wherein said burden conferred by synthesis of said product, when said first and second essential gene in the cell are operably linked to their native promoters, has a fitness cost measured as a percent reduction in the maximum exponential phase growth rate of the microbial production cell selected from among > 5 %, >10%, >15%, >20%, >25%, >35% and > 45 % measured relative to a corresponding non-producing microbial cell.

3. The microbial production cell according to any one of claims 1 or 2, wherein the cell is: i. a bacterium belonging to a genus selected from among Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Proprionibacterium, Bacteroides, and Bifidobacterium; or

II. a yeast belonging to a genus selected from among Saccharomyces, Kluyveromyces, Candida, Pichia, Komagataella, Cryptococcus, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces and Schizosaccharomyces; or iii. a filamentous fungus selected from among Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma Thermomyces, Streptomyces and Aspergillus.

4. The microbial production cell according to any one of claims 1-3, wherein the first and second essential genes are identical.

5. The microbial production cell according to any one of claims 1-4, wherein the first and second burden-sensing promoters are different.

6. The microbial production cell according to any one of claims 1-5, wherein the first and/or second essential gene is a non-conditional essential gene.

7. The microbial production cell according to any one of claims 1-6, wherein the first and/or second essential gene is operably linked to a synthetic RBS whose sequence is selected to modify the translational strength of the first and/or second essential gene independent of induction of said first and/or second burden-sensing promoter, respectively.

8. The microbial production cell according to any one of claims 1-7, wherein the first and second burden-sensing promoters are selected from among: i. a ribosomal RNA promoter,

II. a promoter upregulated by oxyR or a homolog thereof, iii. a promoter comprising a UPR element upregulated by HAC1 or a homolog thereof, and iv. a DNA damage-sensing promoter.

9. The microbial production cell according to any one of claims 1-8, wherein the cell is characterized by an increased product yield after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 generations of cell division from a single cell, as compared to a parent microbial production cell lacking said first and second essential genes operably linked to said first and second burden-sensing promoters, respectively.

10. The microbial production cell according to any one of claims 1-9, wherein the cell is characterized by an increased product yield of at least 10, 25, 50, or 80% following at least 50 generations of cell division from a single cell as compared to a parent microbial production cell lacking said first and second essential genes operably linked to said first and second burden-sensing promoters, respectively.

11. A method of product biosynthesis comprising the steps of: i. providing at least one microbial production cell according to any one of claims 1-10,

II. introducing the at least one cell into a cultivation medium comprising substrate for production of said product, ill. recovering said product.

12. The method according to claim 11, further comprising a step of isolating the product and/or formulating the product into a composition, such as a nutritional, pharmaceutical, cosmetic, detergent, lubricant, or fuel composition.

13. A method of product biosynthesis according to claim 11 or 12, wherein the product is selected from among: an organic acid, terpenoid, isoprenoid, polyketide, alcohol, sugar, vitamin, aldehyde, carboxylic acid, fatty acid, amino acid, peptide, enzyme (such as an amylase, lipase, protease, barnase, p- galactosidase, crystal protein, cutinase, PETase, laccase and carbohydrate active enzyme (such as a xylanase, lichenase, cellulase, lytic polysaccharide monooxoygenase, and pectase)), a therapeutic protein and a precursor thereof (such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal- and polyclonal-antibody, single-fragment antibody and nanobody), a protein naturally found in eggs (such as ovalbumin), a milk protein (such as casein, lactadherin, lactoferrin), secreted immunoglobulin A and G, secretory components.

14. Use of a first and a second essential gene operably linked to a first and a second burden-sensing promoter, respectively, to enhance product yield of a cultured population of microbial production cells arising from a single cell following at least 50 generations; wherein said first burden-sensing promoter is heterologous with respect to said first essential gene, and said second burden-sensing promoter is heterologous with respect to said second essential gene; wherein synthesis of the product confers a burden on said cell; and wherein expression of said first essential gene is up-regulated when said first burden-sensing promoter is induced by said burden relative to a basal level expression of said first essential gene when said first burden-sensing promoter is not induced, and expression of said second essential gene is up-regulated when said second burden-sensing promoter is induced by said burden relative to a basal level expression of said second essential gene when said second burden-sensing promoter is not induced.

15. Use of said first and second essential gene operably liked to said first and second burden-sensing promoter, respectively, according to claim 14, wherein product yield is enhanced by at least 10, 25, 50, or 80% following at least 50 generations of cell division from a single cell as compared to a parent microbial production cell lacking said first and second essential genes operably linked to said first and second burden-sensing promoters, respectively.

16. Use of a microbial production cell according to any one of claims 1-10 for producing a biosynthetic product, such as organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, amino acids, peptides, enzymes (such as amylases, lipases, proteases, barnases, p-galactosidases, crystal proteins, cutinases, PETases, laccases and carbohydrate active enzymes (such as xylanases, lichenases, cellulases, lytic polysaccharide monooxoygenases, and pectases)), therapeutic proteins and precursors thereof (such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, single-fragment antibodies), proteins naturally found in eggs (such as ovalbumin), milk proteins (such as caseins, lactadherins, lactoferrin), secreted immunoglobulin A and G, secretory components, nanobodies.